Diagnosis, prognosis and identification of potential therapeutic targets of multiple myeloma based on gene expression profiling

ABSTRACT

Gene expression profiling is a powerful tool that has varied utility. It enables classification of multiple myeloma into subtypes and identifying genes directly involved in disease pathogensis and clinical manifestation. The present invention used gene expression profiling in large uniformly treated population of patients with myeloma to identify genes associated with poor prognosis. It also demonstrated that over-expression of CKS1B gene, mainly due to gene amplification that was determined by Fluorescent in-situ hybridization to impart a poor prognosis in multiple myeloma. It is further contemplated that therapeutic strategies that directly target CKS1B or related pathways may represent novel, and more specific means of treating high risk myeloma and may prevent its secondary evolution.

RELATED APPLICATIONS

This application is a continuation of patent application U.S. Ser. No.12/587,383 filed Oct. 6, 2009, which is a continuation-in-part of patentapplication U.S. Ser. No. 11/110,209, filed Apr. 20, 2005, now U.S. Pat.No. 7,935,679, which is a continuation-in-part of patent applicationU.S. Ser. No. 10/931,780, filed Sep. 1, 2004, now U.S. Pat. No.7,371,736, which is a continuation-in-part of patent application U.S.Ser. No. 10/454,263, filed Jun. 4, 2003, now U.S. Pat. No. 7,308,364,which is a continuation-in-part application of patent application U.S.Ser. No. 10/409,004, filed Apr. 8, 2003, now U.S. Pat. No. 7,894,992,which is a continuation-in-part of patent application U.S. Ser. No.10/289,746, filed Nov. 7, 2002, now U.S. Pat. No. 7,668,659, whichclaims benefit of provisional patent applications U.S. Ser. No.60/403,075, filed Aug. 13, 2002, U.S. Ser. No. 60/355,386, filed Feb. 8,2002, and U.S. Ser. No. 60/348,238, filed Nov. 7, 2001. The entireteachings of U.S. Ser. No. 12/587,383 are incorporated herein byreference.

FEDERAL FUNDING LEGEND

This invention was made with government support under grants CA097513and CA055819 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

INCORPORATION BY REFERENCE OF MATERIAL IN ASCII TEXT FILE

This application incorporates by reference the Sequence Listingcontained in the following ASCII text file being submitted concurrentlyherewith:

File name: 46411000015SeqList.txt; created May 22, 2013, 1.09 KB insize.

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates generally to the field of cancer research.More specifically, the present invention relates to gene expressionprofiling of a large, uniformly-treated population of patients withmyeloma to identify genes associated with poor prognosis.

Description of the Related Art

Multiple myeloma (MM) is a uniformly fatal tumor of terminallydifferentiated plasma cells (PCs) that home to and expand in the bonemarrow. Although initial transformation events leading to thedevelopment of multiple myeloma are thought to occur at a post-germinalcenter stage of development as suggested by the presence of somatichypermutation of IGV genes, progress in understanding the biology andgenetics of multiple myeloma has been slow.

Multiple myeloma cells are endowed with a multiplicity of anti-apoptoticsignaling mechanisms that account for their resistance to currentchemotherapy and thus the ultimately fatal outcome for most patients.While aneuploidy by interphase fluorescence in situ hybridization (FISH)and DNA flow cytometry are observed in >90% of cases, cytogeneticabnormalities in this typically hypoproliferative tumor are informativein only about 30% of cases and are typically complex, involving onaverage 7 different chromosomes. Given this “genetic chaos” it has beendifficult to establish correlations between genetic abnormalities andclinical outcomes. Only recently has chromosome 13 deletion beenidentified as a distinct clinical entity with a grave prognosis.However, even with the most comprehensive analysis of laboratoryparameters, such as β2-microglobulin (β2M), C-reactive protein (CRP),plasma cell labeling index (PCLI), metaphase karyotyping, and FISH, theclinical course of patients afflicted with multiple myeloma can only beapproximated because no more than 20% of clinical heterogeneity can beaccounted for. Thus, there are distinct clinical subgroups of multiplemyeloma, and modern molecular tests may provide help in identifyingthese entities.

Monoclonal gammopathy of undetermined significance (MGUS) and multiplemyeloma are the most frequent forms of monoclonal gammopathies.Monoclonal gammopathy of undetermined significance is the most commonplasma cell dyscrasia with an incidence of up to 10% of population overage 75. The molecular basis of monoclonal gammopathy of undeterminedsignificance and multiple myeloma are not very well understood and it isnot easy to differentiate these two disorders. Diagnosis of multiplemyeloma or monoclonal gammopathy of undetermined significance isidentical in ⅔ of cases using classification systems that are based on acombination of clinical criteria such as the amount of bone marrowplasmocytosis, the concentration of monoclonal immunoglobulin in urineor serum, and the presence of bone lesions. Especially in early phasesof multiple myeloma, differential diagnosis is associated with a certaindegree of uncertainty.

Furthermore, in the diagnosis of multiple myeloma, clinician mustexclude other disorders in which a plasma cell reaction may occur. Theseother disorders include rheumatoid arthritis, connective tissuedisorders, and metastatic carcinoma where the patient may haveosteolytic lesions associated with bone metastases. Therefore, giventhat multiple myeloma is thought to have an extended latency andclinical features are recognized many years after development of themalignancy, new molecular diagnostic techniques are needed fordifferential diagnosis of multiple myeloma, e.g., monoclonal gammopathyof undetermined significance versus multiple myeloma, or recognition ofvarious subtypes of multiple myeloma.

Additionally, although this malignancy of B-cell origin initiallyresides in the bone marrow, it can transform into an aggressive diseasewith an abnormal karyotype, increased proliferation, elevated LDH andextra-medullary manifestations (Barlogie, B et al., 2001). Specificmolecular genetic lesions and tumor cell-stroma interaction influencethe clinical course and response to therapy (Kuehl, W. M. et al., 2002;Shaughnessy, J et al., 2003; Hideshima, T et al., 2004; Fonseca, R. etal., 2004). Although complete responses can be obtained in more than 40%of patients with high-dose therapy, survival varies widely from fewmonths to more than 15 years (Attal, M. et al., 2003; Barlogie, B. etal., 2004). High-risk disease is best captured by abnormal metaphasecytogentics, present in one-third of newly diagnosed patients andreflecting high proliferative capacity of the malignant disease(Shaughnessy, J. et al., 2003).

Global gene expression profiling has emerged as a powerful tool forclassifying disease subtypes and developing robust prognostic models inleukemia and lymphoma (Shipp, M. A. et al., 2002; Yeoh, E. J. et al.,2002; Rosenwald, A. et al., 2002; Bullinger, L. et al., 2004; Valk, P.J. et al., 2004). In myeloma, this technology helped identify genesdirectly involved in disease pathogenesis and clinical manifestation(Zhan, F et al., 2002; Zhan, F. et al., 2003; Tarte, K et al., 2003;Tian, E. et al., 2003).

Thus, the prior art is deficient in methods for identifying genesassociated with poor prognosis in patients with myeloma. The presentinvention fulfills this long-standing need and desire in the art.

SUMMARY OF THE INVENTION

Bone marrow plasma cells from 74 patients with newly diagnosed multiplemyeloma, 5 patients with monoclonal gammopathy of undeterminedsignificance (MGUS), and 31 normal volunteers (normal plasma cells) werepurified by CD138⁺ selection. Gene expression of purified plasma cellsand 7 multiple myeloma cell lines were profiled using high-densityoligonucleotide microarrays interrogating ˜6,800 genes. Usinghierarchical clustering analysis, normal and multiple myeloma plasmacells were differentiated and four distinct subgroups of multiplemyeloma (MM1, MM2, MM3 and MM4) were identified. The gene expressionpatterns of MM1 were similar to that of normal plasma cells andmonoclonal gammopathy of undetermined significance, whereas MM4 wassimilar to multiple myeloma cell lines. Clinical parameters linked topoor prognosis such as abnormal karyotype (p=0.0003) and high serumβ2-microglobulin levels (p=0.0004) were most prevalent in MM4. Genesinvolved in DNA metabolism and cell cycle control were overexpressed inMM4 as compared to MM1.

Using chi square and Wilcoxon rank sum tests, 120 novel candidatedisease genes that discriminated between normal and malignant plasmacells (p<0.0001) were identified. Many of these candidate genes areinvolved in adhesion, apoptosis, cell cycle, drug resistance, growtharrest, oncogenesis, signaling and transcription. In addition, a totalof 156 genes, including FGFR3 and CCND1, exhibited highly elevated(“spiked”) expression in at least 4 of the 74 multiple myeloma cases(range of spikes: 4 to 25). Elevated expression of FGFR3 and CCND1 werecaused by translocation t(4;14)(p16;q32) or t(11;14)(q13;q32).

Additionally, multivariate stepwise discriminant analysis was used toidentify a minimum subset of genes whose expression was intimatelyassociated with malignant features of multiple myeloma. Fourteen geneswere defined as predictors that were able to differentiate plasma cellsof multiple myeloma patients from normal plasma cells with a high degreeof accuracy, and 24 genes were identified as predictors that are able todifferentiate distinct subgroups of multiple myeloma (MM1, MM2, MM3 andMM4) described herein.

Furthermore, it was also demonstrated that multiple myeloma could beplaced into a developmental schema parallel to that of normal plasmacell differentiation. Based on gene expression profiling, the MM4, MM3and MM2 subgroups described above were found to have similarity withtonsil B cells, tonsil plasma cells and bone marrow plasma cellsrespectively. These data suggested that the enigmatic multiple myelomais amendable to a gene expression/development stage-based classificationsystem.

Thus, gene expression profiling using DNA microarray and hierarchicalclustering analysis could be used to classify subgroups of multiplemyeloma, identify genes with differential expression in subsets ofmultiple myeloma patients, and identify potential therapeutic targetsfor multiple myeloma. For example, multiple myeloma or subgroups ofmultiple myeloma can be diagnosed based on the expression of a group of14 genes or a group of 24 genes respectively. Additionally, multiplemyeloma can be diagnosed based on the expression levels of 15 genes thatclassify patients into 7 subgroups of myeloma.

In another aspect of the present invention, multiple myeloma can betreated using methods that involve inhibiting or enhancing expression ofgenes that are found to be over-expressed or downregulated respectivelyin multiple myeloma patients as disclosed herein. Additionally, multiplemyeloma can be classified based on developmental stage by comparing geneexpression profiling between multiple myeloma cells and normal B orplasma cells.

In one aspect of the present invention, there is provided a method ofidentifying genes associated with a phenotype of interest in apopulation. This method comprises isolating plasma cells fromindividuals within the population. Subsequently, oligonucleotidemicroarrays may be used to profile gene expression in the cells.Further, the gene expression profiles in the cells are correlated withthe phenotype of interest. Thus, the genes associated with the phenotypeof interest in the population are identified. The present inventionprovides a method of diagnosing an individual with myeloma. This methodcomprises (a) obtaining a bone marrow sample from the individual and (b)determining an amplification of a gene on chromosome 1q21 or a deletionof chromosome 13 in the sample, thereby diagnosing the individual withmyeloma. The present invention further provides a method of identifyingan individual having high-risk myeloma. This method comprises (a)obtaining a bone marrow sample from the individual and (b) determiningan amplification of a gene on chromosome 1q21, where the amplificationof the gene increases the risk of developing myeloma, therebyidentifying the individual having high-risk myeloma. The presentinvention also provides a method of screening drugs useful in treatinghigh-risk myeloma. Such a method comprises contacting a samplecomprising CKS1B with a drug and determining the inhibitory effect ofthe drug on amplification, over-expression or activity of CKS1B gene,thereby screening for drugs useful in treating high-risk myeloma.

An alternative approach to the method of screening drugs useful intreating high-risk myeloma would comprise contacting a sample comprisingCKS1B with a drug and determining the inhibitory effect of the drug onamplification, over-expression or activity of CKS1B protein. The presentinvention still further provides a method of treating an individualhaving high-risk myeloma. Such a method comprises administering to theindividual a compound that inhibits amplification, over-expression oractivity of CKS1B gene. An alternative approach to the method oftreating an individual having high-risk myeloma would compriseadministering to the individual a compound that inhibits amplification,over-expression or activity of CKS1B protein.

Other and further aspects, features, and advantages of the presentinvention will be apparent from the following description of thepresently preferred embodiments of the invention. These embodiments aregiven for the purpose of disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows cluster-ordered data table. The clustering is presentedgraphically as a colored image. Along the vertical axis, the analyzedgenes are arranged as ordered by the clustering algorithm. The geneswith the most similar patterns of expression are placed adjacent to eachother. Likewise, along the horizontal axis, experimental samples arearranged; those with the most similar patterns of expression across allgenes are placed adjacent to each other. Both sample and gene groupingscan be further described by following the solid lines (branches) thatconnect the individual components with the larger groups. The color ofeach cell in the tabular image represents the expression level of eachgene, with red representing an expression greater than the mean, greenrepresenting an expression less than the mean, and the deeper colorintensity representing a greater magnitude of deviation from the mean.FIG. 1B shows amplified gene cluster showing genes downregulated in MM.Most of the characterized and sequence-verified cDNA-encoded genes areknown to be immunoglobulins. FIG. 1C shows cluster enriched with geneswhose expression level was correlated with tumorigenesis, cell cycle,and proliferation rate. Many of these genes were also statisticallysignificantly upregulated in multiple myeloma (χ² and WRS test) (seeTable 5). FIG. 1D shows dendrogram of hierarchical cluster. 74 cases ofnewly diagnosed untreated multiple myeloma, 5 monoclonal gammopathy ofundetermined significance, 8 multiple myeloma cell lines, and 31 normalbone marrow plasma cell samples clustered based on the correlation of5,483 genes (probe sets). Different-colored branches represent normalplasma cell (green), monoclonal gammopathy of undetermined significance(blue arrow), multiple myeloma (tan) and multiple myeloma cell lines(brown arrow). FIG. 1E shows dendrogram of a hierarchical clusteranalysis of 74 cases of newly diagnosed untreated multiple myeloma alone(clustergram note shown). Two major branches contained two distinctcluster groups. The subgroups under the right branch, designated MM1(light blue) and MM2 (blue) were more related to the monoclonalgammopathy of undetermined significance cases in FIG. 1D. The twosubgroups under the left branch, designated MM3 (violet) and MM4 (red)represent samples that were more related to the multiple myeloma celllines in FIG. 1D.

FIG. 2 shows two-dimensional hierarchical cluster analysis ofexperimental expression profiles and gene behavior of 30 EDG-MM. Bcells, tonsil and bone marrow plasma cells, and multiple myeloma (MM)samples were analyzed using a cluster-ordered data table. The tonsil Bcell, tonsil plasma cell, bone marrow plasma cell samples are indicatedby red, blue, and golden bars respectively. The nomenclature for the 74MM samples is as indicated in Zhan et al. (2002a). Along the verticalaxis, the analyzed genes are arranged as ordered by the clusteringalgorithm. The genes with the most similar patterns of expression areplaced adjacent to each other. Both sample and gene groupings can befurther described by following the solid lines (branches) that connectthe individual components with the larger groups. The tonsil B cellcluster is identified by the horizontal red bar. The color of each cellin the tabular image represents the expression level of each gene, withred representing an expression greater than the mean, green representingan expression less than the mean, and the deeper color intensityrepresenting a greater magnitude of deviation from the mean.

FIG. 3 shows two-dimensional hierarchical cluster analysis ofexperimental expression profiles and gene behavior of 50 LDG-MM1 genes.Genes are plotted along the vertical axis (right side), and experimentalsamples are plotted along the top horizontal axis by their similarity.The tonsil plasma cell cluster is identified by a horizontal blue bar.Tonsil B cell, tonsil plasma cell, and bone marrow plasma cell samplesare indicated as in FIG. 18.

FIG. 4 shows two-dimensional hierarchical cluster analysis ofexperimental expression profiles and gene behavior of 50 LDG-MM2 genes.Genes are plotted along the vertical axis (right side), and experimentalsamples are plotted along the top horizontal axis by their similarity.The bone marrow plasma cell cluster is identified by a horizontal goldenbar. Tonsil B cell, tonsil plasma cell, and bone marrow plasma cellsamples are indicated as in FIG. 18.

FIG. 5 shows variation in expression of proliferation genes revealssimilarities between tonsil B cells and MM4. The data are shown asboxplot of Kruskal-Wallis test values. The seven groups analyzed (tonsilB cells, tonsil plasma cells, bone marrow plasma cells, and geneexpression defined subgroups MM1, MM2, MM3, and MM4) are distributedalong the x-axis and the natural log transformed average difference isplotted on the y axis. EZH2; p=7.61×10⁻¹¹; KNSL1, p=3.21×10⁻⁸; PRKDC,p=2.86×10⁻¹¹; SNRPC, p=5.44×10⁻¹²; CCNB1, p=2.54×10⁻⁸; CKS2,p=9.49×10⁻¹¹; CKS1, p=5.86×10⁻⁹; PRIM1, p=4.25×10⁻⁵.

FIG. 6A-B show that CKS1B expression by myeloma plasma cells is variableand high levels define a high-risk myeloma entity. FIG. 6A shows boxplots of log base 2-transformed Affymetric signal plotted on the y-axiswith respect to 351 cases according to the quartile expression levelsx-axis). FIG. 6B shows Kaplan-Meier plots of overall survival thatrevealed inferior outcome among the 88 patients with 4^(th) quartileexpression levels of CKS1B compared to the remaining 263 patients withquartile 1-3 expression levels.

FIG. 7A-D show that increased CKS1B expression is related to CKS1B DNAcopy number and degree of DNA amplification is linked to poor survival.FIG. 7A shows metaphase fluorescence in situ hybridization analysis ofCKS1B at 1q21 (red signal) and ASPM at 1q31 (green signal) performed onplasma cells from a patient with myeloma. The tandem duplications ofCKS1B (arrows) and the greater degree of amplification of 1q21 werecompared to 1q31. FIG. 7B shows box plots of log base 2-transformedAffymetrix signal (y-axis) by CKS1B amplification (N=197). In box plots,the top, bottom and middle lines of each box correspond to the 75^(th)percentile (top quartile), 25^(th) percentile (bottom quartile) and50^(th) percentile (median) respectively and, the whiskers extend to thenearest point not beyond 1.5 times the inter-quartile range, withobservations beyond these indicated by individual lines. A Wilcoxon ranksum test was used to compare Signal across copy number categories. FIG.7C shows Kaplan-Meier plot of overall survival in the validation cohortthat depicts inferior outcomes among the 89 patients with CKS1Bamplification compared to the remaining 135, as determined by interphasefluorescence in situ hybridization. FIG. 7D shows the Kaplan-Meier plotas shown in 28C for the combined sample of 421 patients.

FIG. 8 shows that CKS1B expression increases in relapsed myeloma. Thisfigure shows CKS1B signal for 32-paired diagnosis and relapse arrays.The quartile 4 reference line is taken from the complete (N=351) sampleof arrays at diagnosis. A majority of samples showed increasedexpression at relapse and the most dramatic changes were in those withquartile1 to quartile3 expression levels at diagnosis. For aWelch-modified paired t-test was used to compare log-scale Signal atdiagnosis and relapse.

FIGS. 9A-E show that CKS1B mRNA is correlated with nuclear proteinlevels and inversely correlated with p27^(Kip1). FIG. 9A shows CKS1B andFIG. 9B shows CDKN1B (p27^(Kip1)) gene expression signal in 1000 unitincrements plotted on the y-axis. Primary myelomas with CKS1B expressionin quartile 1 (n=13) and quartile 4 (n=14) and myeloma cell lines (n=7)were grouped and plotted from left to right along the X-axis. Each barrepresents a sample and the height indicates the level of geneexpression in the sample. FIGS. 9C-E show Western Blot analysis ofnuclear protein extracts for CKS1B (FIG. 9C), phos-thr-187-p27^(Kip1)(FIG. 9D) and Histone 1A (FIG. 9E) (loading control) respectively fromaliquots of same plasma cells used in FIG. 9A and FIG. 9B. Samples areordered from left to right in the exact same order in all panels. Thereis a high degree of correlation between CKS1B gene expression andprotein expression. CDKN1B (p27^(Kip1)) gene expression is notcorrelated with CKS1B gene expression, protein levels or p27^(Kip1)protein levels. However, there is a strong inverse correlation betweenCKS1B protein levels and p27^(Kip1) protein levels. Uniform histone 1Aprotein levels indicate equal protein loading across all samples.

FIG. 10 shows the chromosome locations of AHCYL1 (1p13), CKS1b(1q21),Rb1(D13S31) and 13qTel(D13S285).

FIG. 11 shows a typical results of FISH on a bone marrow specimen of apatient with newly diagnosed multiple myeloma.

FIG. 12A-D show the Kaplan-Meier analysis of overall survival (OS) bychromosome 1q21 (CKS1b) abnormalities. FIG. 12A show for all treatmentprotocols. FIG. 12B show for Total Therapy 3 (TT3). FIG. 12C show forTotal Therapy 2 (TT2) without thalidomide. FIG. 12D show for TotalTherapy 2 (TT2) with thalidomide.

FIG. 13A-D show the Kaplan-Meier analysis of even free survival (EFS) bychromosome 1q21 (CSK1b) abnormalities. FIG. 13A show for all treatmentprotocols. FIG. 13B show for Total Therapy 3 (TT3). FIG. 13C show forTotal Therapy 2 (TT2) without thalidomide. FIG. 13D show for TotalTherapy 2 (TT2) with thalidomide.

FIG. 14A-D show the Kaplan-Meier analysis of overall survival (OS) bychromosome 1p13 (AHCYL1) abnormalities. FIG. 14A show for all treatmentprotocols. FIG. 14B show for Total Therapy 3 (TT3). FIG. 14C show forTotal Therapy 2 (TT2) without thalidomide. FIG. 14D show for TotalTherapy 2 (TT2) with thalidomide.

FIG. 15A-D show the Kaplan-Meier analysis of even free survival (EFS) bychromosome 1p13 (AHCYL1) abnormalities. FIG. 15A show for all treatmentprotocols. FIG. 15B show for Total Therapy 3 (TT3). FIG. 15C show forTotal Therapy 2 (TT2) without thalidomide. FIG. 15D show for TotalTherapy 2 (TT2) with thalidomide.

FIG. 16A-D show the Kaplan-Meier analysis of overall survival (OS) bychromosome 13q14 (Rb1) abnormalities. FIG. 16A show for all treatmentprotocols. FIG. 16B show for Total Therapy 3 (TT3). FIG. 16C show forTotal Therapy 2 (TT2) without thalidomide. FIG. 16D show for TotalTherapy 2 (TT2) with thalidomide.

FIG. 17A-D show the Kaplan-Meier analysis of even free survival (EFS) bychromosome 13q14 (Rb1) abnormalities. FIG. 17A show for all treatmentprotocols. FIG. 17B show for Total Therapy 3 (TT3). FIG. 17C show forTotal Therapy 2 (TT2) without thalidomide. FIG. 17D show for TotalTherapy 2 (TT2) with thalidomide.

FIG. 18A-D show the Kaplan-Meier analysis of overall survival (OS)between 1q21 (CKS1b) and 1p13 (AHCYL1) abnormalities. FIG. 18A shows forall treatment protocols. FIG. 18B shows for Total Therapy 3 (TT3). FIG.18C shows for Total Therapy 2 (TT2) without thalidomide. FIG. 18D showsfor Total Therapy 2 (TT2) with thalidomide.

FIG. 19A-D show the Kaplan-Meier analysis of event free survival (EFS)between 1q21 (CKS1b) and 1p13 (AHCYL1) abnormalities. FIG. 19A shows forall treatment protocols. FIG. 19B shows for Total Therapy 3 (TT3). FIG.19C shows for Total Therapy 2 (TT2) without thalidomide. FIG. 19D showsfor Total Therapy 2 (TT2) with thalidomide.

FIG. 20A-D show the Kaplan-Meier analysis of overall survival (OS)between 1q21 (CKS1b) and 13q14 (Rb1) abnormalities. FIG. 20A shows forall treatment protocols. FIG. 20B shows for Total Therapy 3 (TT3). FIG.20C shows for Total Therapy 2 (TT2) without thalidomide. FIG. 20D showsfor Total Therapy 2 (TT2) with thalidomide.

FIG. 21A-D show the Kaplan-Meier analysis of event free survival (EFS)between 1q21 (CKS1b) and 13q14 (Rb1) abnormalities. FIG. 21A shows forall treatment protocols. FIG. 21B shows for Total Therapy 3 (TT3). FIG.21C shows for Total Therapy 2 (TT2) without thalidomide. FIG. 21D showsfor Total Therapy 2 (TT2) with thalidomide.

FIG. 22A-F show the Kaplan-Meier analysis of survival outcomes of 1q21(CKS1b) abnormalities based on gene copy numbers of 1, 2, 3 or 4. FIG.22A-C show overall survival rates. FIG. 22D-F show event free survivalrates.

FIG. 23A-F show the Kaplan-Meier analysis of survival outcomes of 1q21(CKS1b) abnormalities based on gene copy numbers of 2 and 1, 3 orgreater. FIGS. 23A-C show overall survivability rates. FIG. 23D-F showevent free survivability rates.

FIG. 24A-F show the Kaplan-Meier analysis of survival outcomes of 1p13(AHCYL1) abnormalities based on gene copy numbers of 1 and 2 or greater.FIG. 24A-C show overall survivability rates. FIGS. 24D-F show event freesurvivability rates.

FIG. 25A-F show the Kaplan-Meier analysis of survival outcomes of 13q14(D13S31) abnormalities based on gene copy numbers of 1 and 2 or greater.FIG. 25A-C show overall survivability rates. FIG. 25D-F show event freesurvivability rates.

FIG. 26A-B show the Kaplan-Meier analysis of survival outcomes of 1p13(AHCYL1) abnormalities based on gene copy numbers of 1 and 2 or greater.FIG. 26A show the combined overall survivability rates for TT2 and TT3.FIG. 26B show the combined event free survivability rates for TT2 andTT3.

FIG. 27A-B show the Kaplan-Meier analysis of survival outcomes of 13q14(D13S31) abnormalities based on gene copy numbers of 1 and 2 or greater.FIG. 27A show the combined overall survivability rates for TT2 and TT3.FIG. 27B show the combined overall survivability rates for TT2 and TT3.

FIG. 28A-B show the Kaplan-Meier analysis of survival outcomes of 1q21(CKS1b) abnormalities based on gene copy numbers of 1, 2, 3, or 4. FIG.28A show the overall survivability rates for TT2 and TT3. FIG. 28B showthe overall survivability rates for TT2 and TT3.

FIG. 29A-B show the Kaplan-Meier analysis of survival outcomes of 1q21(CKS1b) abnormalities based on gene copy numbers of 2 and 1, 3 orgreater. FIG. 29A show combined overall survivability rates for TT2 andTT3. FIG. 29B show combined event free survivability rates for TT2 andTT3.

DETAILED DESCRIPTION OF THE INVENTION

There is now strong evidence that global gene expression profiling canreveal molecular heterogeneity of similar or related hematopoieticmalignancies that have been difficult to distinguish. Genes exhibitingsignificant differential expression between normal and malignant cellscan be used in the development of clinically relevant diagnostics aswell as provide clues into the basic mechanisms of cellulartransformation. In fact, these profiles might even be used to identifymalignant cells even in the absence of any clinical manifestations. Inaddition, biochemical pathways in which the products of these genes actmay be targeted by novel therapeutics.

Both normal and malignant plasma cells were purified to homogeneity frombone marrow aspirates using anti-CD138-based immunomagneticbead-positive selection. Using these cells, the present inventionprovided the first comprehensive global gene expression profiling ofnewly diagnosed multiple myeloma patients and contrasted theseexpression patterns with those of normal plasma cells. Novel candidatemultiple myeloma disease genes were identified and this profiling led todevelopment of a gene-based classification system for multiple myeloma.

Results from hierarchical cluster analysis on multiple myeloma plasmacells and normal plasma cells, as well as the benign plasma celldyscrasia monoclonal gammopathy of undetermined significance andend-stage-like multiple myeloma cell lines revealed normal plasma cellsare unique and that primary multiple myeloma is either like monoclonalgammopathy of undetermined significance or multiple myeloma cell lines.In addition, multiple myeloma cell line gene expression was homogeneousas evidenced by tight clustering in the hierarchical analysis.Similarity of multiple myeloma cell line expression patterns to primarynewly diagnosed forms of multiple myeloma support the validity of usingmultiple myeloma cell lines as models for multiple myeloma.

Four distinct clinical multiple myeloma subgroups (MM1 to MM4) weredistinguished upon hierarchical clustering of multiple myeloma. The MM1subgroup contained samples that were more like monoclonal gammopathy ofundetermined significance, whereas the MM4 subgroup contained samplesmore like multiple myeloma cell lines. The most significant geneexpression patterns differentiating MM1 and MM4 were cell cycle controland DNA metabolism genes, and the MM4 subgroup was more likely to haveabnormal cytogenetics, elevated serum 132M, elevated creatinine, anddeletions of chromosome 13. These are important variables thathistorically have been linked to poor prognosis.

Gene Expression Changes in Multiple Myeloma

The most significant gene expression changes differentiating the MM1 andMM4 subgroups code for activities that clearly implicate MM4 as having amore proliferative and autonomous phenotype. The most significantlyaltered gene in the comparison, TYMS (thymidylate synthase), whichfunctions in the prymidine biosynthetic pathway, has been linked toresistance to fluoropyrimidine chemotherapy and also poor prognosis incolorectal carcinomas. Other notable genes upregulated in MM4 were theCAAX farnesyltransferase gene, FTNA. Farnesyltransferase prenylates RAS,a post translational modification required to allow RAS to attach to theplasma membrane. These data suggest that farnesyltransferase inhibitorsmay be effective in treating patients with high levels of FTNAexpression.

Two other genes coding for components of the proteasome pathway, POH1(26S proteasome-associated pad1 homolog) and UBL1 (ubiquitin-likeprotein 1) were also overexpressed in MM4. Overexpression of POH1confers P-glycoprotein-independent, pleotropic drug resistance tomammalian cells. UBL1, also known as sentrin, is involved in manyprocesses including associating with RAD51, RAD52, and p53 proteins inthe double-strand repair pathway; conjugating with RANGAP1 involved innuclear protein import; and importantly for multiple myeloma, protectingagainst both Fas/Apo-1 (TNFRSF6) or TNFR1-induced apoptosis. In contrastto normal plasma cells, more than 75% of multiple myeloma plasma cellsexpress abundant mRNA for the multidrug resistance gene,lung-resistance-related protein (MVP). These data are consistent withprevious reports showing expression of MVP in multiple myeloma is a poorprognostic factor. Given the uniform development of chemotherapyresistance in multiple myeloma, the combined overexpression of POH1 andMVP may have profound influences on this phenotype. The deregulatedexpression of many genes whose products function in the proteasomepathway may be used in pharmacogenomic analysis in determining theefficacy of proteasome inhibitors like PS-341 (MillenniumPharmaceuticals, Cambridge, Mass.).

Another significantly upregulated gene in MM4 was the single strandedDNA-dependent ATP-dependent helicase (G22P1), which is also known asKu70 autoantigen. The DNA helicase II complex, made up of p70 and p80,binds preferentially to fork-like ends of double-stranded DNA in a cellcycle-dependent manner. Binding to DNA is thought to be mediated by p70and dimerization with p80 forms the ATP-dependent DNA-unwinding enzyme(helicase II) and acts as the regulatory component of a DNA-dependentprotein kinase (DNPK) which was also significantly upregulated in MM4.The involvement of helicase II complex in DNA double-strand breakrepair, V(D)J recombination, and notably chromosomal translocations hasbeen proposed. Another upregulated gene was the DNA fragmentation factor(DFFA). Caspase-3 cleaves DFFA-encoded 45 kD subunit at two sites togenerate an active factor that produces DNA fragmentation duringapoptosis signaling. In light of the many blocks to apoptosis inmultiple myeloma, DFFA activation could result in DNA fragmentation,which in turn would activate the helicase II complex that may facilitatechromosomal translocations. It is of note that abnormal karyotypes, andthus chromosomal translocations, are associated with the MM4 subgroupwhich tended to overexpress these two genes.

Hence, it was demonstrated that direct comparison of gene expressionpatterns in multiple myeloma and normal plasma cells identified novelgenes that could represent fundamental changes associated with malignanttransformation of plasma cells.

Progression of multiple myeloma as a hypoproliferative tumor is thoughtto be linked to a defect in programmed cell death rather than rapid cellreplication. Two genes, prohibitin (PHB) and quiescin Q6 (QSCN6),overexpressed in multiple myeloma are involved in growth arrest.Overexpression of these genes may be responsible for the typically lowproliferation indices seen in multiple myeloma. It is hence conceivablethat therapeutic downregulation of these genes that results in enhancedproliferation could render multiple myeloma cells more susceptible tocell cycle-active chemotherapeutic agents.

The gene coding for CD27, TNFRSF7, the second most significantlyunderexpressed gene in multiple myeloma, is a member of the tumornecrosis factor receptor (TNFR) superfamily that provides co-stimulatorysignals for T and B cell proliferation, B cell immunoglobulin productionand apoptosis. Anti-CD27 significantly inhibits induction of Blimp-1 andJ-chain transcripts which are turned on in cells committed to plasmacell differentiation, suggesting that ligation of CD27 on B cells mayprevent terminal differentiation. CD27 ligand (CD70) preventsIL-10-mediated apoptosis and directs differentiation of CD27⁺ memory Bcells toward plasma cells in cooperation with IL-10. Thus, it ispossible that downregulation of CD27 gene expression in multiple myelomamay block an apoptotic program.

Overexpression of CD47 in multiple myeloma may be related to escape ofmultiple myeloma cells from immune surveillance. Studies have shown thatcells lacking CD47 are rapidly cleared from the bloodstream by splenicred pulp macrophages and CD47 on normal red blood cells prevents thiselimination.

The gene product of DNA methyltransferase 1, DNMT1, overexpressed inmultiple myeloma is responsible for cytosine methylation in mammals andhas an important role in epigenetic gene silencing. In fact, aberranthypermethylation of tumor suppressor genes plays an important role inthe development of many tumors. De novo methylation of p16/INK4a is afrequent finding in primary multiple myeloma. Also, recent studies haveshown that upregulated expression of DNMTs may contribute to thepathogenesis of leukemia by inducing aberrant regional hypermethylation.DNA methylation represses genes partly by recruitment of themethyl-CpG-binding protein MeCP2, which in turn recruits a histonedeacetylase activity. It has been shown that the process of DNAmethylation mediated by Dnmt1 may depend on or generate an alteredchromatin state via histone deacetylase activity. It is potentiallysignificant that multiple myeloma cases also demonstrate significantoverexpression of metastasis-associated 1 (MTA1) gene. MTA1 wasoriginally identified as being highly expressed in metastatic cells.MTA1 has more recently been discovered to be one subunit of the NURD(NUcleosome Remodeling and histone Deacetylation) complex which containsnot only ATP-dependent nucleosome disruption activity, but also histonedeacetylase activity. Thus, over expression of DNMT1 and MTA1 may havedramatic effects on repressing gene expression in multiple myeloma.

Oncogenes activated in multiple myeloma included ABL and MYC. Althoughit is not clear whether ABL tyrosine kinase activity is present inmultiple myeloma, it is important to note that overexpression of abl andc-myc results in accelerated development of mouse plasmacytomas. Thus,it may be more than a coincidence that multiple myeloma cellssignificantly overexpresses MYC and ABL.

Chromosomal translocations involving the MYC oncogene and IGH and IGLgenes that result in dysregulated MYC expression are hallmarks ofBurkitt's lymphoma and experimentally induced mouse plasmacytomas;however, MYC/IGH-associated translocations are rare in multiple myeloma.Although high MYC expression was a common feature in our panel ofmultiple myeloma, it was quite variable, ranging from little or noexpression to highly elevated expression. It is also of note that theMAZ gene whose product is known to bind to and activate MYC expressionwas significantly upregulated in the MM4 subgroup. Given the importantrole of MYC in B cell neoplasia, it is speculated that overexpression ofMYC, and possibly ABL, in multiple myeloma may have biological andpossibly prognostic significance.

EXT1 and EXT2, which are tumor suppressor genes involved in hereditarymultiple exostoses, heterodimerize and are critical in the synthesis anddisplay of cell surface heparan sulfate glycosaminoglycans (GAGs). EXT1is expressed in both multiple myeloma and normal plasma cells. EXT2L wasoverexpressed in multiple myeloma, suggesting that a functionalglycosyltransferase could be created in multiple myeloma. It is of notethat syndecan-1 (CD138/SDC1), a transmembrane heparan sulfateproteoglycan, is abundantly expressed on multiple myeloma cells and,when shed into the serum, is a negative prognostic factor. Thus,abnormal GAG-modified SDC1 may be important in multiple myeloma biology.The link of SDC1 to multiple myeloma biology is further confirmed by therecent association of SDC1 in the signaling cascade induced by WNTproto-oncogene products. It has been showed that syndecan-1 (SDC1) isrequired for Wnt-1-induced mammary tumorigenesis. Data disclosed hereinindicated a significant downregulation of WNT10B in primary multiplemyeloma cases. It is also of note that the WNT5A gene and the FRZB gene,which codes for a decoy WNT receptor, were also marginally upregulatedin newly diagnosed multiple myeloma. Given that WNTs represent a novelclass of B cell regulators, deregulating the expression of these growthfactors (WNT5A, WNT10B) and their receptors (e.g., FRZB) and genesproducts that modulate receptor signaling (e.g., SDC1) may be importantin the genesis of multiple myeloma.

Genes identified by the present invention that show significantlyup-regulated or downregulated expression in multiple myeloma arepotential therapeutic targets for multiple myeloma. Over-expressed genesmay be targets for small molecules or inhibitors that decrease theirexpression. Methods and materials that can be used to inhibit geneexpression, e.g. small drug molecules, anti-sense oligo, or antibodywould be readily apparent to a person having ordinary skill in this art.On the other hand, under-expressed genes can be replaced by gene therapyor induced by drugs.

Gene Profiles Defining Disease Subgroups

A multivariate stepwise discriminant analysis was used to identify aminimum subset of genes whose expression was intimately associated withmalignant features of multiple myeloma. By applying linear regressionanalysis to the top 50 differentially expressed genes, 14 genes weredefined as predictors that are able to differentiate multiple myelomafrom normal plasma cells with a high degree of accuracy. When the modelwas applied to a validation group consisting of 118 multiple myeloma, 6normal plasma cells and 7 cases of monoclonal gammopathy of undeterminedsignificance (MGUS), an accuracy of classification of more than 99% wasachieved. Importantly, 6 of the 7 MGUS cases were classified as multiplemyeloma, indicating that MGUS has gene expression features ofmalignancy. Thus the altered expression of 14 genes out of over 6,000genes interrogated are capable of defining multiple myeloma. Similarmultivariate discriminant analysis also identified a set of 24 genesthat can distinguish between the four multiple myeloma subgroupsdescribed above.

In addition to identifying genes that were statistically differentbetween normal plasma cells and multiple myeloma plasma cells, thepresent invention also identified genes, like FGFR3 and CCND1, thatdemonstrate highly elevated “spiked” expression in subsets of multiplemyelomas. Patients with elevated expression of these genes can havesignificant distribution differences among the four gene expressioncluster subgroups. For example, FGFR3 spikes are found in MM1 and MM2whereas spikes of IFI27 are more likely to be found in MM3 and MM4.Highly elevated expression of the interferon-induced gene IFI27 may beindicative of viral infection, either systemic or specifically withinthe plasma cells from these patients. Correlation analysis has shownthat IFI27 spikes are significantly linked (Pearson correlationcoefficient values of 0.77 to 0.60) to elevated expression of 14interferon-induced genes, including MX1, MX2, OAS1, OAS2, IFIT1, IFIT4,PLSCR1, and STAT1. More recent analysis of a large population ofmultiple myeloma patients (N=280) indicated that nearly 25% of allpatients had spikes of the IFI27 gene. It is of interest to determinewhether or not the IFI27 spike patients who cluster in the MM4 subgroupare more likely to have a poor clinical course and to identify thesuspected viral infection causing upregulation of this class of genes.In conclusion, spiked gene expression may also be used in thedevelopment of clinically relevant prognostic groups.

Finally, the 100% coincidence of spiked FGFR3 or CCND1 gene expressionwith the presence of t(4;14)(p14;q32) or t(11;14)(q13;q32)translocations, as well as the strong correlations between proteinexpression and gene expression represent important validations of theaccuracy of gene expression profiling and suggests gene expressionprofiling may eventually supplant the labor intensive and expensiveclinical laboratory procedures, such as cell surface markerimmunophenotyping and molecular and cellular cytogenetics.

In another embodiment, a feature-subset selection was used to extractgenes relevant to specific myeloma subtypes. In this regard,multivariate stepwise discriminant analysis was applied and identified15 genes that could correctly separate tissue samples into sevensubtypes. Examining the expression of the 15 genes thus identified notonly would provide important new insights into the diagnosis andpathogenesis of these myeloma subtypes but also may pinpoint usefultargets against which novel therapeutic agents could be developed.

Comparison of Multiple Myeloma with Normal Plasma Cell Development

Further, it was also shown that multiple myeloma can could be placedinto a developmental schema parallel to that of normal plasma celldifferentiation. Global gene expression profiling revealed distinctchanges in transcription associated with human plasma celldifferentiation. Hierarchical clustering analyses with 4866 genessegregated tonsil B cells, tonsil plasma cells, and bone marrow plasmacells. Combining χ² and Wilcoxon rank sum tests, 359 previously definedand novel genes significantly (p<0.0005) discriminated tonsil B cellsfrom tonsil plasma cells, and 500 genes significantly discriminatedtonsil plasma cells from bone marrow plasma cells. Genes that weredifferentially expressed in the tonsil B cell to tonsil plasma celltransition were referred as “early differentiation genes” (EDGs) andthose differentially expressed in the tonsil plasma cell to bone marrowplasma cell transition were referred as “late differentiation genes”(LDGs). One-way ANOVA was then applied to EDGs and LDGs to identifystatistically significant expression differences between multiplemyeloma (MM) and tonsil B cells (EDG-MM), tonsil plasma cells (LDG-MM1),or bone marrow plasma cells (LDG-MM2).

Hierarchical cluster analysis revealed that 13/18 (p=0.00005) MM4 cases(a putative poor-prognosis subtype) clustered tightly with tonsil Bcells. The other groups (MM1, 2 and 3) failed to show such associations.In contrast, there was tight clustering between tonsil plasma cells and14/15 (p=0.00001) MM3, and significant similarities were found betweenbone marrow plasma cells and 14/20 (p=0.00009) MM2 cases. MM1 showed nosignificant linkage with the normal cell types studied. In addition,XBP1, a transcription factor essential for plasma cell differentiation,exhibited a significant, progressive reduction in expression from MM1 toMM4, consistent with developmental-stage relationships. Therefore,global gene expression patterns linked to late-stage B celldifferentiation confirmed and extended a global gene expression-definedclassification system of multiple myeloma, suggesting that multiplemyeloma represents a constellation of subtypes of disease with uniqueorigins.

Identification of Genes that could be Useful as Diagnostic, Prognosticand Targets in Myeloma

The present invention also identified genes that could be useful asdiagnostic, prognostic and potential targets in myeloma. 70 genes whoseexpression was significantly correlated with disease-related survivalwas identified by performing gene expression analysis of highly purifiedplasma cells from newly diagnosed myeloma patients; 30% of these genesmapped to chromosome 1. Increased expression of 1q genes and reducedexpression of 1p genes were consistent with cytogenetic data of frequent1q gains and 1p loses in myeloma karyotypes (Nilsson et al., 2003;Gutierrez et al., 2004). Tandem duplications and jumping translocationsinvolving 1q21, caused by decondensation of pericentromericheterochromatin are features of end stage disease (Sawyer et al., 2005;Sawyer et al., 1998; Le Baccon et al., 2001).

Over expression and amplification of CKS1B, mapping to 1q21 was linkedto poor prognosis in newly diagnosed myeloma. Its role in controllingSCF^(Skp2)-mediated ubiquitinylation and proteasomal degradation of thecyclin-dependent kinase inhibitor p27^(Kip1) made it an attractivecandidate gene. CKS1B protein levels were correlated with geneexpression and both inversely correlated with p27^(Kip1) protein levels.Investigations in S. cerevisiae demonstrated an essential role of cks1in promoting mitosis by modulating the transcriptional activation ofCDC20 (Morris et al., 2003). A strong correlation between CKS1B andCDC20 expression (r=0.78; p<0.0001) was observed in the presentinvention, consistent with CKS1B promoting mitosis by regulating CDC20expression in human cells. Therefore, the results obtained in thepresent invention demonstrate that gene dosage-related increase in CKS1Bexpression led to enhanced degradation of p27^(Kip1) and possiblyactivation of CDC20.

In context of the recently recognized prognostically relevant geneticsubgroups, CKS1B hyper-activation was less frequent in cases withhyperdiploid and normal karyotypes; one-third of those withCCND1-translocations had high CKS1B levels and upto two-thirds ofhigh-risk entities, MAF, MAFB and hyperdiploidy displayed CKS1Bhyperactivation (Table 6A). In addition to conferring a poor prognosisamong newly diagnosed patients, CKS1B over-expression and amplificationwere common at relapse in patients lacking these features at diagnosis.Hence, it will be important to determine whether CKS1B amplificationemerges in all subgroups and when present, portends rapid diseaseprogression and death.

CKS1B gene amplification along with abnormal metaphase cytogenetics andchromosome 13 deletion accounted for almost 40% of the observed survivalvariability, underscored that myeloma risk was best assessed by studyingmolecular and cellular genetics. It is therefore recommended toroutinely apply such studies, performed on a single bone marrow samplefor appropriate patient stratification in therapeutic trial design.

The impact of new agents such as bortezomib and thalidomide and itsderivatives will be profound if their clinical efficacy is extended togenetically defined high-risk myeloma. Since CKS1B directly orindirectly interacts with ubiquitin ligases and/or the proteasome toregulate multiple cell cycle checkpoints (Pagano and Benmaamar, 2003),it is contemplated that new therapeutic strategies that directly targetCKS1B or related pathways might represent novel and more specific meansof treating de novo high-risk myeloma and might prevent its secondaryevolution.

Further, fluorescence in-situ hybridization (FISH) tests for 1q21amplification and 13 deletion represented a better test for riskassessment that existed. Since the expression of CKS1B highly correlatedwith the gene copy number and the risk of death increased with theincrease in gene copy number, this test could also be used to stagepatients at diagnosis, detect residual diseases, predict recurrence andprogression of the disease.

In summary, the method of gene expression profiling for multiple myelomacomprises applying nucleic acid samples of isolated plasma cells derivedfrom individuals with or without multiple myeloma to a DNA microarray,and performing hierarchical clustering analysis on data obtained fromthe microarray to classify the individuals into distinct subgroups suchas the MM1, MM2, MM3 and MM4 subgroups disclosed herein. Gene expressionprofiling will also identify genes with elevated expression in subsetsof multiple myeloma patients or genes with significantly differentlevels of expression in multiple myeloma patients as compared to normalindividuals. These genes are potential therapeutic targets for multiplemyeloma.

In another embodiment, a group of genes will be identified that candistinguish between normal plasma cells and plasma cells of multiplemyeloma. Nucleic acid samples of isolated plasma cells derived fromindividuals with or without multiple myeloma were applied to a DNAmicroarray, and hierarchical clustering analysis was performed on dataobtained from the microarray. Genes with statistically significantdifferential expression patterns were identified, and linear regressionanalysis was used to identify a group of genes that is capable ofaccurate discrimination between normal plasma cells and plasma cells ofmultiple myeloma. This analysis can also identify a group of genes thatis capable of accurate discrimination between subgroups of multiplemyeloma.

The expression levels of a group of 14 genes thus identified could beused for diagnosis of multiple myeloma. Significant differentialexpression of these genes would indicate that such individual hasmultiple myeloma or a subgroup of multiple myeloma. Gene expressionlevels can be examined at nucleic acid level or protein level accordingto methods well known to one of skill in the art.

An another embodiment, comprises a method of diagnosis for multiplemyeloma based on examining the expression levels of a group of genescomprising CST6, RAB7L1, MAP4K3, HRASLS2, TRAIL, IG, FGL2, GNG11, MCM2,FLJ10709, CCND1, MAF, MAFB, FGFR3, and MMSET. CCND1, MAF, MAFB, FGFR3,and MMSET expression can be determined by and are correlated withchromosomal translocation such as t(4;14)(p21;q32), t(14;16)(q32;q23),t(14;20)(q32;q13), and t(11;14)(q13;q32). Gene expression levels of thisgroup of genes would classify an individual into one of 7 groups ofmyeloma (groups 1-7). Individual in myeloma groups 1, 2, 3 and 6 wouldhave poor prognosis compared to individual in myeloma groups 4, 5 and 7.Group 1 is defined by downregulation of CST6, RAB7L1, MAP4K3, HRASLS2,GNG11, MCM2 and FLJ10709, and overexpression of TRAIL, IG and FGFL2.Group 2 is defined by downregulation of CST6, RAB7L1, MAP4K3, HRASLS2,IG, FGFL2, MCM2 and FLJ10709, and overexpression of TRAIL and GNG11.Group 3 is defined by overexpression of CCND1, or translocationt(11;14)(q13;q32). Group 4 is defined by downregulation of CST6, RAB7L1,MAP4K3, HRASLS2, IG, FGFL2, and GNG11, and overexpression of TRAIL, MCM2and FGFL2. Group 5 is defined by overexpression of MAF or MAFB, ortranslocation t(14;16)(32;q23) or t(14;20)(q32;q13). Group 6 is definedby overexpression of CST6, RAB7L1, MAP4K3, and HRASLS2, anddownregulation of TRAIL. Group 7 is defined by overexpression of FGFR3or MMSET, or translocation t(4;14)(p21;q32).

In yet another embodiment are methods of treatment for multiple myeloma.Such methods involve inhibiting expression of one of the genes orincreasing expression of one of the genes. Methods of inhibiting orincreasing gene expression such as those using anti-senseoligonucleotide or antibody are well known to one of skill in the artInhibiting gene expression can be achieved through RNA interferenceusing so called siRNA. Gene expression enhancement might be through genetherapy.

In still yet another embodiment is a method of developmental stage-basedclassification for multiple myeloma. Nucleic acid samples of isolated Bcells and plasma cells derived from individuals with or without multiplemyeloma were applied to a DNA microarray, and hierarchical clusteringanalysis performed on data obtained from the microarray will classifythe multiple myeloma cells according to the developmental stages ofnormal B cells and plasma cells. In general, normal B cells and plasmacells are isolated from tonsil, bone marrow, mucoal tissue, lymph nodeor peripheral blood.

In another embodiment, there is a method of controlling bone loss in anindividual by inhibiting the expression of the DKK1 gene (accessionnumber NM012242). In general, DKK1 expression can be inhibited byanti-sense oligonucleotides or anti-DKK1 antibodies. In anotherembodiment, bone loss can be controlled by a pharmacological inhibitorof DKK1 protein. Preferably, the individual having bone loss may havemultiple myeloma, osteoporosis, post-menopausal osteoporosis ormalignancy-related bone loss that is caused by breast cancer metastasisor prostate cancer metastasis.

The present invention is drawn to a method of identifying genesassociated with a phenotype of interest in a population, comprising:isolating cells from individuals within the population, profiling geneexpression in the cells using oligonucleotide microarrays, correlatingthe gene expression profiles in the cells with the phenotype ofinterest, thereby identifying genes associated with the phenotype ofinterest in the population. Generally, the phenotype of interest isdisease-related survival, response to a drug.

The present invention is also drawn to a method of diagnosing anindividual with myeloma, comprising the steps of: (a) obtaining a bonemarrow sample from the individual, and (b) determining an amplificationof a gene on chromosome 1q21 or a deletion of chromosome 13 in thesample, thereby diagnosing the individual with myeloma. This methodcomprises predicting relapse or progression of the disease in theindividual based on the extent of amplification of the gene onchromosome 1q21, where an increase in the amplification of the geneindicates relapse or progression of the disease in the individual.Specifically, the amplified gene is CKS1B.

The present invention is further drawn to a method of identifying anindividual having high-risk myeloma comprising the steps of: (a)obtaining a bone marrow sample from the individual; and (b) determiningan amplification of a gene on chromosome 1q21, wherein the amplificationof the gene increases the risk of developing myeloma, therebyidentifying the individual having high-risk myeloma. This method furthercomprises: predicting survival of the individual having high-riskmyeloma by detecting an amplification of the gene on chromosome 1q21,where the amplification of the gene on chromosome 1q21 is associatedwith high-incidence of myeloma-related death. Specifically, theamplified gene is CKS1B.

The present invention is still further drawn to a method of screeningfor drugs useful in treating high-risk myeloma, comprising: contacting asample comprising CKS1B with a drug, and determining the inhibitoryeffect of the drug on amplification, over-expression or activity ofCKS1B gene, thereby screening for drugs useful in treating high-riskmyeloma.

Alternatively, the present invention is also drawn to a method ofscreening for drugs useful in treating high-risk myeloma, comprising;contacting a sample comprising CKS1B with a drug, and determining theinhibitory effect of the drug on amplification, over-expression oractivity of the CKS1B protein, thereby screening for drugs useful intreating high-risk myeloma. As is known to one skilled in the art thatsuch a drug will also target components that are either upstream ordownstream of this gene or protein in a cell cycle-related pathway.

The present invention is further drawn to a method of treating anindividual having a high-risk myeloma, comprising: administering to theindividual a compound that inhibits amplification, over-expression oractivity of CKS1B gene. Examples of such compounds although not limitedto include compounds such as a peptide nucleic acid (PNA), RNA-mediatedinterference. Alternatively, the present invention is also drawn to amethod of treating an individual having a high-risk myeloma, comprising:administering to the individual a compound that inhibits amplification,over-expression or activity of CKS1B protein. Examples of such compoundsalthough not limited to include compounds such as an antibody, a CKS1Bantisense RNA or a small molecule inhibitor.

The following examples are given for the purpose of illustrating variousembodiments of the invention and are not meant to limit the presentinvention in any fashion. One skilled in the art will appreciate readilythat the present invention is well adapted to carry out the objects andobtain the ends and advantages mentioned, as well as those objects, endsand advantages inherent herein. Changes therein and other uses which areencompassed within the spirit of the invention as defined by the scopeof the claims will occur to those skilled in the art.

Example 1 Cell Isolation and Analysis

Samples for the following studies included plasma cells from 74 newlydiagnosed cases of multiple myeloma, 5 subjects with monoclonalgammopathy of undetermined significance, 7 samples of tonsil Blymphocytes (tonsil BCs), 11 samples of tonsil plasma cells (tonsilPCs), and 31 bone marrow PCs derived from normal healthy donor. Multiplemyeloma cell lines (U266, ARP1, RPMI-8226, UUN, ANBL-6, CAG, and H929(courtesy of P. L. Bergsagel) and an Epstein-Barr virus(EBV)-transformed B-lymphoblastoid cell line (ARH-77) were grown asrecommended (ATCC, Chantilly, Va.).

Tonsils were obtained from patients undergoing tonsillectomy for chronictonsillitis. Tonsil tissues were minced, softly teased and filtered. Themononuclear cell fraction from tonsil preparations and bone marrowaspirates were separated by a standard Ficoll-Hypaque gradient(Pharmacia Biotech, Piscataway, N.J.). The cells in the light densityfraction (S.G.≦1.077) were resuspended in cell culture media and 10%fetal bovine serum, RBC lysed, and several PBS wash steps wereperformed. Plasma cell isolation was performed with anti-CD138immunomagnetic bead selection as previously described (Zhan et al.,2002). B lymphocyte isolation was performed using directly conjugatedmonoclonal mouse anti-human CD19 antibodies and the AutoMacs automatedcell sorter (Miltenyi-Biotec, Auburn, Calif.).

For cytology, approximately 40,000 purified tonsil BC and PC mononuclearcells were cytocentrifuged at 1000×g for 5 min at room temperature. Formorphological studies, the cells were immediately processed by fixingand staining with DiffQuick fixative and stain (Dade Diagnostics,Aguada, PR). For immunofluorescence, slides were treated essentially asdescribed (Shaughnessy et al., 2000). Briefly, slides were air-driedovernight, then fixed in 100% ethanol for 5 min at room temperature andbaked in a dry 37° C. incubator for 6 hr. The slides were then stainedwith 100 μl of a 1:20 dilution of goat anti-human-kappa immunoglobulinlight chain conjugated with 7-amino-4-methylcourmarin-3-acitic acid(AMCA) (Vector Laboratories, Burlingame, Calif.) for 30 min in ahumidified chamber. After incubation, the slides were washed two timesin 1×PBS+0.1% NP-40 (PBD). To enhance the AMCA signal, the slides wereincubated with 100 μl of a 1:40 dilution of AMCA-labeledrabbit-anti-goat IgG antibody and incubated for 30 min at roomtemperature in a humidified chamber. Slides were washed 2 times in1×PBD. The slides were then stained with 100 μl of a 1:100 dilution ofgoat anti-human-lambda immunoglobulin light chain conjugated with FITC(Vector Laboratories, Burlingame, Calif.) for 30 min in a humidifiedchamber; the slides were washed two times in 1×PBD. Then the slides werestained with propidium iodide at 0.1 μg/ml in 1×PBS for 5 min, washed in1×PBD, and 10 μl anti-fade (Molecular Probes, Eugene, Oreg.) was addedand coverslips were placed. Cytoplasmic immunoglobulin lightchain-positive PCs were visualized using an Olympus BX60epi-fluorescence microscope equipped with appropriate filters. Theimages were captured using a Quips XL genetic workstation (Vysis,Downers Grove, Ill.).

Both unpurified mononuclear cells and purified fractions from tonsilBCs, tonsil PCs, and bone marrow PCs were subjected to flow cytometricanalysis of CD marker expression using a panel of antibodies directlyconjugated to FITC or PE. Markers used in the analysis includedFITC-labeled CD20, PE-labeled CD38, FITC-labeled or ECD-labeled CD45,PE- or PC5-labeled CD138 (Beckman Coulter, Miami, Fla.). For detectionof CD138 on PCs after CD138 selection, we employed an indirect detectionstrategy using a FITC-labeled rabbit anti-mouse IgG antibody (BeckmanCoulter) to detect the mouse monoclonal anti-CD138 antibody BB4 used inthe immunomagnetic selection technique. Cells were taken after FicollHypaque gradient or after cell purification, washed in PBS, and stainedat 4° C. with CD antibodies or isotype-matched control G1 antibodies(Beckman Coulter). After staining, cells were resuspended in 1×PBS andanalyzed using a Epics XL-MCL flow cytometry system (Beckman Coulter).

Example 2 Preparation of Labeled cRNA and Hybridization to High-DensityMicroarray

Total RNA was isolated with RNeasy Mini Kit (Qiagen, Valencia, Calif.).Double-stranded cDNA and biotinylated cRNA were synthesized from totalRNA and hybridized to HuGeneFL GENECHIP® microarrays (Affymetrix, SantaClara, Calif.), which were washed and scanned according to proceduresdeveloped by the manufacturer. The arrays were scanned using HewlettPackard confocal laser scanner and visualized using Affymetrix 3.3software (Affymetrix). Arrays were scaled to an average intensity of1,500 and analyzed independently.

Example 3 GENECHIP® Microarray Data Analysis

To efficiently manage and mind high-density oligonucleotide DNAmicroarray data, a new data-handling tool was developed. GENECHIP®microarray-derived expression data was stored on an MS SQL Server. Thisdatabase was linked, via an MS Access interface called ClinicalGene-Organizer to multiple clinical parameter databases for multiplemyeloma patients. This Data Mart concept allows gene expression profilesto be directly correlated with clinical parameters and clinical outcomesusing standard statistical software. All data used in the presentanalysis were derived from Affymetrix 3.3 software. GENECHIP® microarray3.3 output files are given (1) as an average difference (AD) thatrepresents the difference between the intensities of thesequence-specific perfect match probe set and the mismatch probe set, or(2) as an absolute call (AC) of present or absent as determined by theGENECHIP® microarray 3.3 algorithm. Average difference calls weretransformed by the natural log after substituting any sample with anaverage difference of <60 with the value 60 (2.5 times the average RawQ). Statistical analysis of the data was performed with softwarepackages SPSS 10.0 (SPSS, Chicago, Ill.), S-Plus 2000 (Insightful Corp.,Seattle, Wash.), and Gene Cluster/Treeview (Eisen et al., 1998).

To differentiate four distinct subgroups of multiple myeloma (MM1, MM2,MM3 and MM4), hierarchical clustering of average linkage clustering withthe centered correlation metric was employed. The clustering was done onthe average difference data of 5,483 genes. Either Chi square (χ²) orFisher's exact test was used to find significant differences betweencluster groups with the AC data. To compare the expression levels, thenon-parametric Wilcoxon rank sum (WRS) test was used. This test uses anull hypothesis that is based on ranks rather than on normallydistributed data. Before the above tests were performed, genes that wereabsent (AC) across all samples were removed; 5,483 genes were used inthe analyses. Genes that were significant (p<0.0001) for both the χ²test and the WRS test were considered to be significantly differentiallyexpressed.

Clinical parameters were tested across multiple myeloma cluster groups.ANOVA test was used to test the continuous variables, and χ² test ofindependence or Fisher's exact test was applied to test discretevariables. The natural log of the average difference data was used tofind genes with a “spiked profile” of expression in multiple myeloma.Genes were identified that had low to undetectable expression in themajority of patients and normal samples (no more than 4 present absolutecalls [PAC]). A total of 2,030 genes fit the criteria of this analysis.The median expression value of each of the genes across all patientsamples was determined. For the i^(th) gene, this value was calledmedgene (i). The i^(th) gene was a “spiked” gene if it had at least 4patient expression values>2.5+medgene (i). The constant 2.5 was based onthe log of the average difference data. These genes that were “spiked”were further divided into subsets according to whether or not thelargest spike had an average difference expression value greater than10,000.

To determine transcriptional changes associated with human plasma celldifferentiation, a total of 4866 genes were scanned across 7 cases eachof tonsil B cells, tonsil plasma cells, and bone marrow plasma cells.The 4866 genes were derived from 6800 by filtering out all controlgenes, and genes not fulfilling the test of Max-Min<1.5 (1.5 being thenatural log of the average difference). The χ² test was used toeliminate genes with absent absolute call (AAC). For example, in thetonsil plasma cell to bone marrow plasma cell comparison, genes with χ²values greater than 3.84 (p<0.05) or having “present” AC (PAC) in morethan half of the samples in each group were retained. In the tonsil Bcell to tonsil plasma cell and tonsil plasma cell to bone marrow plasmacell comparisons, 2662 and 2549 genes were retained as discriminatingbetween the two groups, respectively. To compare gene expression levels,the non-parametric Wilcoxon Rank Sum (WRS) test was used to compare twogroups using natural log transformed AD. The cutoff p value depended onthe sample size, the heterogeneity of the two comparative populations(tonsil B cells, tonsil plasma cells, and bone marrow plasma cellsshowed a higher degree of stability in AD), and the degree ofsignificance. In this analysis, 496 and 646 genes were found to besignificantly (p<0.0005) differentially expressed in the tonsil B cellversus tonsil plasma cell and tonsil plasma cell versus bone marrowplasma cell comparisons, respectively. To define the direction ofsignificance (expression changes being up or down in one group comparedwith the other), the non-parametric Spearman correlation test of the ADwas employed.

Genes that were significantly differentially expressed in the tonsil Bcell to tonsil plasma cell transition were referred as “earlydifferentiation genes” (EDGs) and those differentially expressed in thetonsil plasma cell to bone marrow plasma cell transition were referredas “late differentiation genes” (LDGs). Previously defined and novelgenes were identified that significantly discriminated tonsil B cellsfrom tonsil plasma cells (359 genes) and tonsil plasma cells from bonemarrow plasma cells (500 genes).

To classify multiple myeloma with respect to EDG and LDG, 74 newlydiagnosed cases of multiple myeloma and 7 tonsil B cell, 7 tonsil plasmacell, and 7 bone marrow plasma cell samples were tested for varianceacross the 359 EDGs and 500 LDGs. The top 50 EDGs that showed the mostsignificant variance across all samples were defined as earlydifferentiation genes for myeloma (EDG-MM). Likewise, the top 50 LDGsshowing the most significant variance across all samples were identifiedas late differentiation genes for myeloma-1 (LDG-MM1). Subtracting theLDG-MM1 from the 500 LDG and then applying one-way ANOVA test forvariance to the remaining genes identified the top 50 genes showingsimilarities between bone marrow plasma cells and multiple myeloma.These genes were defined as LDG-MM2.

Hierarchical clustering was applied to all samples using 30 of the 50EDG-MM. A total of 20 genes were filtered out with Max-Min<2.5. Thisfiltering was performed on this group because many of the top 50 EDG-MMshowed no variability across multiple myeloma and thus could not be usedto distinguish multiple myeloma subgroups. A similar clustering strategywas employed to cluster the samples using the 50 LDG-MM1 and 50 LDG-MM2;however, in these cases all 50 significant genes were used in thecluster analysis.

Example 4 RT-PCR and Immunohistochemistry

RT-PCR for the FGFR3MMSET was performed on the same cDNAs used in themicroarray analysis. Briefly, cDNA was mixed with the IGJH2(5′-CAATGGTCACCGTCTCTTCA-3′, SEQ ID No. 1) primer and the MMSET primer(5′-CCTCAATTTCCTGAAATTGGTT-3′, SEQ ID No. 2). PCR reactions consisted of30 cycles with a 58° C. annealing temperature and 1-minute extensiontime at 72° C. using a Perkin-Elmer GeneAmp 2400 thermocycler(Wellesley, Mass.). PCR products were visualized by ethidium bromidestaining after agarose gel electrophoresis.

Immunohistochemical staining was performed on a Ventana ES (VentanaMedical Systems, Tucson, Ariz.) using Zenker-fixed paraffin-embeddedbone marrow sections, an avidin-biotin peroxidase complex technique(Ventana Medical Systems), and the antibody L26 (CD20, Ventana MedicalSystems). Heat-induced epitope retrieval was performed by microwavingthe sections for 28 minutes in a 1.0-mmol/L concentration of citratebuffer at pH 6.0.

Example 5 Interphase FISH

For interphase detection of the t(11;14)(q13;q32) translocation fusionsignal, a LSI IGH/CCND1 dual-color, dual-fusion translocation probe wasused (Vysis, Inc, Downers Grove, Ill.). The TRI-FISH procedure used toanalyze the samples has been previously described. Briefly, at least 100clonotypic plasma cells identified by cIg staining were counted for thepresence or absence of the translocation fusion signal in all samplesexcept one, which yielded only 35 plasma cells. A multiple myelomasample was defined as having the translocation when >25% of the cellscontained the fusion.

Example 6 Hierarchical Clustering of Plasma Cell Gene ExpressionDemonstrates Class Distinction

As a result of 656,000 measurements of gene expression in 118 plasmacell samples, altered gene expression in the multiple myeloma sampleswas identified. Two-dimensional hierarchical clustering differentiatedcell types by gene expression when performed on 5,483 genes that wereexpressed in at least one of the 118 samples (FIG. 1A). The sampledendrogram derived two major branches (FIGS. 1A and 1D). One branchcontained all 31 normal samples and a single monoclonal gammopathy ofundetermined significance case whereas the second branch contained all74 multiple myeloma and 4 monoclonal gammopathy of undeterminedsignificance cases and the 8 cell lines. The multiple myeloma-containingbranch was further divided into two sub-branches, one containing the 4monoclonal gammopathy of undetermined significance and the other the 8multiple myeloma cell lines. The cell lines were all clustered next toone another, thus showing a high degree of similarity in gene expressionamong the cell lines. This suggested that multiple myeloma could bedifferentiated from normal plasma cells and that at least two differentclasses of multiple myeloma could be identified, one more similar tomonoclonal gammopathy of undetermined significance and the other similarto multiple myeloma cell lines.

Hierarchical clustering analysis with all 118 samples together withduplicate samples from 12 patients (plasma cells taken 24 hr or 48 hrafter initial sample) were repeated to show reproducibility of thetechnique and analysis. All samples from the 12 patients studiedlongitudinally were found to cluster adjacent to one another. Thisindicated that gene expression in samples from the same patient weremore similar to each other than they were to all other samples (data notshown).

In addition to the demonstration of reproducibility of clustering notedabove, three microarray analyses were also performed on a single sourceof RNA from one patient. When included in the cluster analysis, thethree samples clustered adjacent to one another. Consistent with themanufacturer's specification, an analysis of the fold changes seen inthe samples showed that <2% of all genes had a >2-fold difference.Hence, these data indicated reproducibility for same samples.

The clustergram (FIG. 1A) showed that genes of unrelated sequence butsimilar function clustered tightly together along the vertical axis. Forexample, a particular cluster of 22 genes, primarily those encodingimmunoglobulin molecules and major histocompatibility genes, hadrelatively low expression in multiple myeloma plasma cells and highexpression in normal plasma cells (FIG. 1B). This was anticipated, giventhat the plasma cells isolated from multiple myeloma are clonal andhence only express single immunoglobulin light-chain and heavy-chainvariable and constant region genes, whereas plasma cells from normaldonors are polyclonal and express many different genes of these twoclasses. Another cluster of 195 genes was highly enriched for numerousoncogenes/growth-related genes (e.g., MYC, ABL1, PHB, and EXT2), cellcycle-related genes (e.g., CDC37, CDK4, and CKS2), and translationmachinery genes (EIF2, EIF3, HTF4A, and TFIIA) (FIG. 1C). These geneswere all highly expressed in MM, especially in multiple myeloma celllines, but had low expression levels in normal plasma cells.

Example 7 Hierarchical Clustering of Newly Diagnosed Multiple MyelomaIdentifies Four Distinct Subgroups

Two-dimensional cluster analysis was performed on the 74 multiplemyeloma cases alone. The sample dendrogram identified two major brancheswith two distinct subgroups within each branch (FIG. 1E). The foursubgroups were designated MM1, MM2, MM3, and MM4 containing 20, 21, 15,and 18 patients respectively. The MM1 subgroup represented the patientswhose plasma cells were most closely related to the monoclonalgammopathy of undetermined significance plasma cells and MM4 were mostlike the multiple myeloma cell lines (see FIG. 1D). These data suggestedthat the four gene expression subgroups were authentic and mightrepresent four distinct clinical entities.

Differences in gene expression across the four subgroups were thenexamined using the χ² and WRS tests (Table 1). As expected the largestdifference was between MM1 and MM4 (205 genes) and the smallestdifference was between MM1 and MM2 (24 genes). Next, the top 30 genesturned on or upregulated in MM4 as compared with MM1 were examined(Table 2). The data demonstrated that 13 of 30 most significant genes(10 of the top 15 genes) were involved in DNA replication/repair or cellcycle control. Thymidylate synthase (TYMS), which was present in all 18samples comprising the MM4 subgroup, was only present in 3 of the 20 MM1samples and represented the most significant gene in the χ² test. TheDNA mismatch repair gene, mutS (E. coli) homolog 2 (MSH2) with a WRS pvalue of 2.8×10⁻⁶ was the most significant gene in the WRS test. Othernotable genes in the list included the CAAX farnesyltransferase (FNTA),the transcription factors enhancer of zeste homolog 2 (EZH2) andMYC-associated zinc finger protein (MAZ), eukaryotic translationinitiation factors (EIF2S1 and EIF2B1), as well as the mitochondrialtranslation initiation factor 2 (MTIF2), the chaperone (CCT4), theUDP-glucose pyrophosphorylase 2 (IUGP2), and the 26Sproteasome-associated pad1 homolog (POH1).

To assess the validity of the clusters with respect to clinicalfeatures, correlations of various clinical parameters across the 4subgroups were analyzed (Table 3). Of 17 clinical variables tested, thepresence of an abnormal karyotype (p=0.0003) and serum 132M levels(p=0.0005) were significantly different among the four subgroups andincreased creatinine (p=0.06) and cytogenetic deletion of chromosome 13(p=0.09) were marginally significant. The trend was to have higher 132Mand creatinine as well as an abnormal karyotype and chromosome 13deletion in the MM4 subgroup as compared with the other 3 subgroups.

TABLE 1 Differences In Gene Expression Among Multiple Myeloma SubgroupsComparison Range of WRS* p Values Number of Genes MM1 vs MM4 .00097 to9.58 × 10⁻⁷ 205 MM2 vs MM4 .00095 to 1.0410⁻⁶ 162 MM3 vs MM4 .00098 to3.7510⁻⁶ 119 MM1 vs MM3 .00091 to 6.2710⁻⁶ 68 MM2 vs MM3 .00097 to1.9810⁻⁵ 44 MM1 vs MM2 .00083 to 2.9310⁻⁵ 24 *Wilcoxon rank sum test.Comparisons are ordered based on the number of significant genes.Comparisons have a WRS p value <0.001.

TABLE 2 The 30 Most Differentially Expressed Genes In A Comparison OfMM1 and MM4 Subgroups Gene MM1 MM4 Chi WRS^(‡) Accession Function Symbol(N = 20) (N = 18) Square p Value D00596 DNA replication TYMS 3 18 24.351.26 × 10⁻⁴ U35835 DNA repair PRKDC 2 17 23.75 4.55 × 10⁻⁶ U77949 DNAreplication CDC6 1 13 15.62 5.14 × 10⁻⁶ U91985 DNA fragmentation DFFA 112 13.38 6.26 × 10⁻⁵ U61145 transcription EZH2 4 15 12.77 1.67 × 10⁻⁴U20979 DNA replication CHAF1A 2 12 10.75 1.10 × 10⁻⁴ U03911 DNA repairMSH2 0 9 10.48 2.88 × 10⁻⁶ X74330 DNA replication PRIM1 0 9 10.48 9.36 ×10⁻⁶ X12517 SnRNP SNR PC 0 9 10.48 5.26 × 10⁻⁶ D85131 transcription MAZ0 9 10.48 1.08 × 10⁻⁵ L00634 farnesyltransferase FNTA 10 18 9.77 7.28 ×10⁻⁵ U21090 DNA replication POLD2 11 18 8.27 8.05 × 10⁻⁵ X54941 cellcycle CKS1 10 17 7.07 1.26 × 10⁻⁴ U62136 cell cycle UBE2V2 13 18 5.574.96 × 10⁻⁶ D38076 cell cycle RANBP1 13 18 5.57 7.34 × 10⁻⁶ X95592unknown C1D^(†) 13 18 5.57 1.10 × 10⁻⁴ X66899 cell cycle EWSR1 14 184.35 1.89 × 10⁻⁴ L34600 translation initiation MTIF2 14 18 4.35 3.09 ×10⁻⁵ U27460 Metabolism IUGP2 15 18 3.22 1.65 × 10⁻⁴ U15009 SnRNP SNRPD315 18 3.22 1.47 × 10⁻⁵ J02645 translation initiation EIF2S1 16 18 2.187.29 × 10⁻⁵ X95648 translation initiation EIF2B1 16 18 2.18 1.45 × 10⁻⁴M34539 calcium signaling FKBP1A 18 18 0.42 1.71 × 10⁻⁵ J04611 DNA repairG22P1 18 18 0.42 7.29 × 10⁻⁵ U67122 anti-apoptosis UBL1 20 18 0.00 7.29× 10⁻⁵ U38846 chaperon CCT4 20 18 0.00 1.26 × 10⁻⁴ U80040 metabolismACO2 20 18 0.00 8.38 × 10⁻⁵ U86782 proteasome POH^(†) 20 18 0.00 5.90 ×10⁻⁵ X57152 signaling CSNK2B 20 18 0.00 7.29 × 10⁻⁵ D87446 unknownKIAA0257 20 18 0.00 1.26 × 10⁻⁵ Accession numbers are GENBANK ® geneticsequence database numbers. ^(†)symbol not HUGO approved. ^(‡)Wilcoxonrank sum test.

TABLE 3 Clinical Parameters Linked To Multiple Myeloma SubgroupsMultiple Myeloma Subgroups Clinical Parameter 1 2 3 4 p value Abnormalcytogenetics 40.0% 5.3% 53.3% 72.2% .00028 Average β2- 2.81 2.73 4.628.81 .00047 microglobulin (mg/L) ANOVA, Chi square, and Fisher's exacttests were used to determine significance.

Example 8 Hierarchical Cluster Analysis with EDG-MM, LDG-MM1, andLDG-MM2 Reveals Developmental Stage-Based Classification of MultipleMyeloma

To identify whether variability in gene expression seen in multiplemyeloma (MM) might be used to discern subgroups of disease, hierarchicalcluster analysis was performed on 74 newly diagnosed MM, 7 tonsil Bcell, 7 tonsil plasma cell, and 7 bone marrow samples using the EDG-MM(FIG. 2), LDG-MM1 (FIG. 3), and LDG-MM2 (FIG. 4). Hierarchicalclustering was applied to all samples using 30 of the 50 EDG-MM. A totalof 20 genes were filtered out with Max-Min<2.5. This filtering wasperformed on this group because many of the top 50 EDG-MM showed novariability across MM and thus could not be used to distinguish MMsubgroups. A similar clustering strategy was employed to cluster thesamples using the 50 LDG-MM1 and 50 LDG-MM2.

The MM samples clustering with the tonsil B cell samples were thenidentified to determine whether the MM cases clustering with tonsil Bcells, or tonsil and bone marrow plasma cells could be correlated withgene expression-defined MM subgroups (Table 4). This data showed that ofthe MM cases clustering tightly with the tonsil B cell samples, 13 of 22were from the MM4 subgroup, accounting for a majority of all MM4 cases(13 of 18 MM4 samples). The LDG-MM defined cluster distribution of geneexpression-defined MM subgroups was dramatically different in that 14 ofthe 28 MM samples clustering with the tonsil plasma cell samples werefrom MM3 subgroup (14 of 15 MM3 samples). LDG-MM2 again showed a strongcorrelation with the MM subgroups in that 14 of the 20 MM cases in thiscluster were from the MM2 subgroup (14 of 21 MM2 cases). Thus, the MM4,MM3, and MM2 subtypes of MM have similarities to tonsil B cells, tonsilplasma cells, and bone marrow plasma cells respectively. MM1 representedthe only subgroup with no strong correlations with normal cellcounterparts tested here, suggesting that this class has uniquecharacteristics yet to be uncovered.

The distribution of the four MM subgroups in the normal cell clustergroups was determined next (Table 5). The results demonstrate thatwhereas all MM3 cases were able to be classified, 6 MM1, 5 MM2, and 3MM4 cases were not clustered with any normal cell group in any of thethree cluster analyses. In all samples capable of being clustered, therewere strong correlations between gene expression-defined subgroups andnormal cell types with the exception of MM1. The data also show that 3MM1, 2 MM2, 4 MM3, and 1 MM4 cases were found to cluster in two groups.No samples were found in three groups and all cases clustering with twonormal classes were always in an adjacent, temporally appropriategroups. P241 was an exception in that it was clustered in the bonemarrow plasma cell and tonsil B cell groups.

Because one of the EDG-MMs was discovered to be cyclin B1 (CCNB1), itwas determined if a panel of proliferation association genes recentlydiscovered to be up-regulated in MM4 could be used to advance andvalidate the classification of MM4 as a so-called tonsil B cell-likeform of MM. Box plots of the expression patterns of CCNB1, CKS1, CKS2,SNRPC, EZH2, KNSL1, PRKDC, and PRIM1 showed significant differencesacross all the groups tested with strong significant correlation betweentonsil B cells and MM4 (FIG. 5). Several important observations weremade in this analysis. For all the genes, with the exception of SNRPC,there was a progressive reduction in expression in the transition fromtonsil B cells to tonsil plasma cells to bone marrow plasma cells. Inaddition, striking correlations were observed with PRIM1 (FIG. 5).Although PRIM1 expression was significantly different across the entiregroup (p=4.25×10⁻⁵), no difference exists between tonsil B cells and MM4(Wilcoxon rank sum [WRS] p=0.1), or between tonsil plasma cells and MM3(WRS p=0.6). Given the important function of several transcriptionfactors in driving and/or maintaining plasma cell differentiation, itwas determined if these factors showed altered expression across thegroups under study. Although other factors showed no significantchanges, XBP1 (FIG. 5) showed an enormous up-regulation between tonsil Bcells and tonsil plasma cells as expected. However, the gene showed areduction in bone marrow plasma cells and a progressive loss across thefour MM subgroups with MM4 showing the lowest level (p=3.85×10⁻¹⁰).

Based on conventional morphological features, plasma cells have beenthought to represent a homogeneous end-stage cell type. However,phenotypic analysis and gene expression profiling disclosed hereindemonstrated that plasma cells isolated from distinct organs can berecognized as belonging to distinct stages of development. Multiplemyeloma plasma cells are derived from the bone marrow and are thought torepresent a transformed counterpart of normal terminally differentiatedbone marrow plasma cells. However, the dramatic differences in survival,which can range from several months to greater than 10 years, suggeststhat multiple myeloma may represent a constellation of several subtypesof disease. Conventional laboratory parameters have not been particularuseful in segregating distinct disease subtypes with sufficientrobustness that would allow adequate risk stratification. In addition,unlike achievements in classifying leukemias and lymphomas based onsimilar nonrandom recurrent chromosomal translocations, the extremekaryotypic heterogeneity of multiple myeloma has made attempts atunderstanding the molecular mechanisms of the disease and classificationprediction virtually impossible.

In studies presented here, it was identified that many EDGs and LDGsexhibit highly variable expression in multiple myeloma, suggesting thatmultiple myeloma might be amenable to a developmental stage-basedclassification. It appears from the results of this study that multiplemyeloma can in fact be classified based on similarities in geneexpression with cells representing distinct stages of B celldifferentiation. This developmental based-system in conjunction with thegene expression-based system reported above represents a criticalaffirmation of the validity of the developmental-based system.

Recent studies provide support for the hypothesis that MM3 represents atonsil plasma cell-like form of the disease. Microarray profiling withthe U95Av2 GENECHIP® microarray on 150 newly diagnosed patients(including the 74 described here) along with an analysis of chromosome13 loss has revealed a significant link between reduced RB1 transcriptswith either monosomy or partial deletions of chromosome 13 (unpublisheddata). In these studies, it was observed that a number of multiplemyeloma cases with or without chromosome 13 deletion had RB1 transcriptsat levels comparable to those seen in normal tonsil plasma cells. FISHanalysis with a bacterial artificial chromosome BAC covering RB1demonstrated that these cases did not have interstitial deletions of theRB1 locus. Given that RB1 was found to be a LDG-MM1, it was determinedif the low levels of RB1 may be linked to tonsil plasma cell-like MM,i.e MM3. Of 35 multiple myeloma cases with RB1 AD values of <1100 (RB1AD value not less than 1100 in 35 normal bone marrow plasma cell samplestested), 74% belonged to the MM3 class. In contrast, of 38 multiplemyeloma cases lacking deletion 13 and having RB1 AD values greater than1100, only 21% belonged to the MM3 subtype.

Although there is a significant link between the cell development-basedclassification and gene expression profiling-based classificationdisclosed herein, there are exceptions in that although as expected themajority of the MM4 cases belonged to the tonsil B cell-clustersubgroup, 5 MM3, 1 MM2, and 3 MM1 cases were also found in this cluster.The recognition that cases within one gene expression-defined subgroupcould be classified in two normal cell defined clusters suggests thesecases may have intermediate characteristics with distinct clinicaloutcomes. It is of interest to determine if the unsupervised geneexpression-based system or developmental stage-based system alone or incombination will allow the creation of robust risk stratificationsystem. This can be tested by allowing sufficient follow-up time on >150uniformly treated multiple myeloma cases in which profiling has beenperformed at diagnosis.

MM1 was the only gene expression-defined subgroup lacking strongsimilarities to any of the normal cell types analyzed in this study. Itis possible that MM1 has similarities to either mucosal-derived plasmacells or peripheral blood plasma cells which has recently been shown torepresent a distinct type of plasma cells. Future studies will be aimedat providing a developmental stage position for this subtype.

The hypoproliferative nature of multiple myeloma, with labeling indexesin the clonal plasma cells rarely exceeding 1%, has lead to thehypothesis that multiple myeloma is a tumor arising from a transformedand proliferative precursor cell that differentiates to terminallydifferentiated plasma cells. It has been shown that there is a bonemarrow B cell population transcribing multiple myeloma plasmacell-derived VDJ joined to IgM sequence in IgG- and IgA-secretingmultiple myelomas. Other investigations have shown that the clonogeniccell in multiple myeloma originates from a pre-switched but somaticallymutated B cell that lacks intraclonal variation. This hypothesis issupported by recent use of single-cell and in situ reversetranscriptase-polymerase chain reaction to detect a high frequency ofcirculating B cells that share clonotypic Ig heavy-chain VDJrearrangements with multiple myeloma plasma cells. Studies have alsoimplicated these precursor cells in mediating spread of disease andaffecting patient survival.

Links of gene expression patterns between subsets of multiple myelomaand cells representing different late stages of B cell differentiationfurther support the above hypothesis in that MM4 and MM3 may haveorigins in a so called “multiple myeloma stem cell”. This hypothesis canbe tested by isolating B cells from tonsils or lymph nodes or peripheralblood of MM3 and MM4 patients, differentiating them into plasma cells invitro using a new method described by Tarte et al. (2002) and thentesting for the presence of an multiple myeloma gene expressionsignature within the differentiated populations. Even if the multiplemyeloma stem cell represents a minority population in the B cells, themultiple myeloma gene expression signature may be recognized, if notwith conventional microarray, then by more sensitive quantitativereal-time. A real time RT-PCR method is envisioned as expression profilemodels using at little as 20 genes that distinguish malignant multiplemyeloma plasma cells from normal plasma cells at an accuracy of 99.5%have been developed.

Regardless of the outcome of these experiments, it is clear that geneexpression profiling has become an extremely powerful tool in evaluatingthe molecular mechanisms of plasma cell differentiation and how theseevents relate to multiple myeloma development and progression, which inturn should provide more rational means of treating this currently fataldisease.

TABLE 4 Distribution of Multiple Myeloma Subgroups in HierarchicalClusters Defined by EDG-MM, LDG-MM1, and LDG-MM2 Genes GeneExpression-Defined MM Subgroups Normal Cell-Defined MM1 MM2 MM3 MM4Cluster (n = 20) (n = 21) (n = 15) n = 18) p EDG-MM 3 1 5 13 .00005 (n =22) LDG-MM1 8 4 14 3 .000008 (n = 29) LDG-MM2 6 14 0 0 .000001 (n = 20)

TABLE 5 Distribution of Gene Expression-Defined Multiple MyelomaSubgroup Cases in Normal Cell Clusters defined by EDG-MM, LDG-MM1, andLDG-MM2 MM1 TBC TPC BPC MM2 TBC TPC BPC MM3 TBC TPC BPC MM4 TBC TPC BPCP026 Y Y P237 Y Y P052 Y Y P034 Y Y P037 Y Y P241 Y Y P098 Y Y P051 YP029 Y Y P079 Y P107 Y Y P057 Y P061 Y P083 Y P158 Y Y P063 Y P066 YP121 Y P119 Y P065 Y P006 Y P144 Y P221 Y P075 Y P120 Y P157 Y P030 YP084 Y P131 Y P171 Y P043 Y P122 Y P002 Y P176 Y P053 Y P127 Y P010 YP213 Y P055 Y P154 Y P067 Y P215 Y P138 Y P187 Y P226 Y P251 Y P155 YP199 Y P025 Y P250 Y P163 Y P255 Y P082 Y P222 Y P239 Y P054 Y P085 P103Y P175 Y P101 Y P099 P202 Y P056 P001 P015 P091 P016 P048 P168 P036 P124P118 P212 P249 MM1, MM2, MM3, MM4, and PXXX represent geneexpression-defined subgroups and patient identifiers, respectively. Yindicates that the case was found in the normal cell-defined cluster.Cases in italics were not found to cluster with any normal cell type.Some cases were found to cluster with two normal cell types. TBC, tonsilB cells; TPB, tonsil plasma cells; BPC, bone marrow plasma cells.

Example 9 Gene Expression Profiling to Identify Genes that could beUseful as Diagnostic, Prognostic and Potential Targets in Myeloma

Global gene expression profiling identified genes directly involved inpathogenesis and classification of myeloma. Next, this profiling wasused to identify genes whose abnormal expression may cause an aggressivephenotype of myeloma. (a) Subjects: 668 newly diagnosed patients withsymptomatic or progressive multiple myeloma, which included 2 cycles ofblood stem cell-supported high-dose melphalan (200 mg/m²) were enrolledin this study. A subset of 575 patients with available geneticmeasurements constituted sample for this analysis. Their medianfollow-up was 30 months. There were 185 progression or death vents and128 deaths. Patient characteristic were as follows: 20% were 65 years orolder, 31% had beta-2-microglobulin levels>=4 mg/L, 55% had C-reactiveprotein levels>=4 mg/L; 22% presented with hemoglobin values<=10 g/dL,10% with creatinine values>=2 mg/dL; LDH was elevated (>=190 IU/L) in15%; cytogenetic abnormalities were detected in 33%. (b) Gene ExpressionProfiling: Gene expression profiling was performed using U133Plus2.0oligonucleotide microarrays as described (Tian, et al., 2003) for aconsecutive 351 patient subset. The expression value ‘Signal’ for eachprobe set was derived from the MAS5.01 software (Affymetrix, SantaClara, Calif.). Median follow-up for survival in this subset was 22months and there were 98 events and 64 deaths. (c) Fluorescence In SituHybridization: Bacterial artificial chromosomes specific for CKS1B at1q21 (RP11-307C12) and ASPM (RP11-32D17) at 1q31 were purchased fromBAC/PAC Resources (Oakland, Calif.) and directly labeled with Spectrumgreen or Spectrum red (Vysis Inc, Downers Grove, Ill.). Metaphasefluorescence in situ hybridization was performed as previously described(Sawyer et al., 2005). The probes were confirmed to map to the 1q21 and1q31 bands using metaphase spreads from normal human lymphocytes. Triplecolor interphase fluorescence in situ hybridization analyses ofchromosomes 13 (RB) and 1q21 (CKS1B) copy number (Shaughnessy et al.,2000) were performed in a subset of 421 patients (145 events and 100deaths, follow-up of 31 months); deletion 13q was scored positivewhen >=80% of clonal cells exhibited loss of a signal at 13q14 asdescribed previously (McCoy et al., 2003). Of these 421 patients, 197were among those with microarrays and 224 were not. (d) WesternBlotting: Nuclear protein was isolated from an aliquot of CD138 enrichedplasma cells that were also analyzed by microarray. Western Blotting wascarried out using the WesternBreeze® Chemiluminiscent Immunodetectionprotocol as described (Invitrogen, Carlsbad, Calif.). The antibodies toCKS1B and phospho-thr-187-p27^(Kip1) were purchased from Zymedlaboratories Inc., (South San Francisco, Calif.) and anti-Histone 1A waspurchased from Upstate Biotechnology (Charlottesville, Va.). (e)Statistical Analysis: False discovery rates methods (Storey andTibshirani, 2003) were used to adjust for multiple comparisons of theAffymetrix probe set Signals for the 351 microarrays. For eachAffymetrix Signal, log rank tests for the equality of disease-relatedsurvival distributions were performed separately for quartile 1 vsquartiles 2 through 4 (to identify under-expressed genes) and quartile 4vs quartiles 1 through 3 (to identify over-expressed genes). A falsediscovery rate cut-off of 2.5% was applied to each list and a total of70 probe sets were retained. Extreme quartile membership (Q1 or Q4) wasassociated with a higher incidence of disease-related death in allretained probe sets. All other EFS and survival outcomes in thisanalysis were overall (i.e. not disease-related). The Kaplan-Meiermethod was used to estimate event-free and overall survivaldistributions and log rank tests were used to test for their equalityacross groups. Chi-square tests and Fisher's exact test were used totest for the independence of categories. Proportional hazards regressionwas used to compare the effect of CKS1B amplification to other variablesand the proportions of observed heterogeneity (i.e. R²) were computed(O'Quigley and Xu, 2001). The statistical packages R version 2.0 (RDevelopment Core Team, 2004) and SPLUS 6.1 (Insightful Corp., 2002) wereused for this analysis. (f) Definition of Genetic Subgroups: Nearly 50%of the newly diagnosed myelomas contain one of five recurrentchromosomal translocations that result in the hyperactivation of MAF,MAFB, FGFR3/MMSET, CCND3, CCND1 (Kuehl, W. M. et al., 2002) withdivergent prognoses (Fonseca, R. et al., 2004), detectable as “spiked”expression by microarray analysis (Zhan, F. et al., 2003). Geneticsubgroups were classified within the context of metaphase cytogentics ashaving normal karyotypes (originating in normal hematopoetic cells incase of hypoproliferative myeloma) or as having hyperdiploid,hypodiploid or “other” cytogenetic abnormalities. “Other” is defined asan abnormal metaphase karyotype, i.e. structural and/or numeric changes,with a diploid modal chromosome number. Thus, non-translocation entitieswere defined by metaphase rather than interphase cytogenetics. (g)Fluorescence In Situ Hybridization-based “CKS1B Amplification Index”: Aconventional, laboratory defined cutoff of 20% for the proportion ofclonal plasma cells with 3 signals or >=4 signals was used for tests ofassociation between expression and amplification (FIG. 2B and Table 3)and for validation of the association between amplification and overallsurvival (FIGS. 2C-D). Hypothesizing that 2 or more extra copies wouldconfer additional risk compared to 1 extra copy, the multivariateanalysis of overall survival (Table 5A) estimated separate effect sizesfor the 3 signal proportion and the >=4 signal proportion. The index wasdefined as a weighted sum: (0.34*% 3 copies+0.66*%>=4 copies)/0.66,where the weights were the log-scale hazard ratio estimates for the twopercentages, scaled to the effect of a unit increase in the proportionwith >=4 signals. The estimated log-scale hazard ratio corresponding toa ne unit difference in the proportion with >=4 signals is nearly twiceas large as that for 3 signals (i.e. 0.66/0.34=1.94). The index is 0 forpatients with <=2 signals in 100% f clonal cells and 100 for patientswith >=4 signals in 100% of clonal cells. The full range was observed inthese patients. A cut-off for the index of >=46 minimized the unadjustedlog rank P-value for survival in the 421 patient subset, however, allcutoffs between 3 and 88 had P<0.003.

Example 10 Results of Global Gene Profiling

To define de novo high-risk multiple myeloma, the gene expressionprofiles in purified myeloma plasma cells were correlated withdisease-related and overall survival in 351 newly diagnosed patientstreated with 2 cycles of high-dose melphalan. Using log rank tests, 70genes were identified for which fourth or first quartile membership wascorrelated with a high incidence of disease-related death (Table 6).

TABLE 6A Quartile 4FDR 2.5% gene probe sets-rank correlations with 1q21amplification index, CKS1B and PC labeling index and adjusted P-valuesfor associations with overall survival. CKS1B Adjusted RankAmplification CKS1B PCLI Survival (Q4) Chromosome Probe set Symbol Indexr ^(†) r^(‡) r* P-value^(a) 1 8q21.13 202345_s_at NA 0.20 0.22 0.001 2Xp22.2-p22.1 1555864_s_at NA 0.34 0.47 0.007 3 5p15.33 204033_at TRIP130.19 0.45 0.20 0.001 4 1q22 206513_at AIM2 0.15 0.13 0.089 5 2p24.11555274_a_at SELI 0.28 0.31 0.001 6 21q22.3 211576_s_at SLC19A1 0.170.23 0.007 7 3p21.3 204016_at LARS2 −0.18 0.002 8 1q43 1565951_s_at OPN30.36 0.36 0.007 9 1q31 219918_s_at ASPM 0.36 0.64 0.17 0.010 10 12q15201947_s_at CCT2 0.23 0.43 0.13 0.004 11 16p13.3 213535_s_at UBE2I 0.380.022 12 20q13.2-q13.3 204092_s_at STK6 0.31 0.51 0.19 0.044 131p36.33-p36.21 213607_x_at FLJ13052 0.150 14 xq12-q13 208117_s_atFLJ12525 0.34 0.006 15 17q25 210334_x_at BIRC5 0.20 0.36 0.14 0.110 163q27 204023_at NA 0.29 0.62 0.16 0.072 17 1q21.2 201897_s_at CKS1B 0.501.00 0.15 0.007 18 19q13.11-q13.12 216194_s_at CKAP1 0.24 0.38 0.001 191q21 225834_at MGC57827 0.39 0.66 0.23 0.140 20 19q13.12 238952_x_atDKFZp779O175 0.11 0.009 21 17p13.3 200634_at PFN1 0.30 0.41 0.002 2219p13.2 208931_s_at ILF3 0.22 0.22 0.220 23 1q22 206332_s_at IFI16 0.300.32 0.13 0.003 24 7p14-p13 220789_s_at TBRG4 0.13 0.17 0.009 2510p11.23 218947_s_at PAPD1 0.31 0.30 0.150 26 8q24 213310_at EIF2C2 0.280.37 0.031 27 3q12.1 224523_s_at MGC4308 0.17 0.24 0.14 0.038 281p36.3-p36.2 201231_s_at ENO1 0.23 <0.001 29 18q12.1 217901_at DSG2 0.150.005 30 6q22 226936_at NA 0.15 0.52 0.17 0.027 31 8q24.3 58696_atEXOSC4 0.20 0.330 32 1q21-q25 200916_at TAGLN2 0.47 0.52 0.120 33 3q21201614_s_at RUVBL1 0.16 0.14 0.023 34 16q22-q24 200966_x_at ALDOA 0.210.28 0.001 35 2p25.1 225082_at CPSF3 0.39 0.073 36 1q43 242488_at NA0.18 0.27 0.14 0.090 37 3q12.3 243011_at MGC15606 0.27 0.004 38 22q13.1201105_at LGALS1 0.31 0.051 39 3p25-p24 224200_s_at RAD18 0.17 0.41 0.140.040 40 20p11 222417_s_at SNX5 0.085 41 1q21.2 210460_s_at PSMD4 0.580.59 0.13 0.067 42 12q24.3 200750_s_at RAN 0.22 0.40 0.056 431pter-q31.3 206364_at KIF14 0.41 0.57 0.25 0.019 44 7p15.2 201091_s_atCBX3 0.14 0.20 0.16 0.150 45 12q22 203432_at TMPO 0.32 0.59 0.18 0.00746 17q24.2 221970_s_at DKFZP586L0724 0.27 0.47 0.081 47 11p15.3-p15.1212533_at WEE1 0.20 0.54 0.13 0.056 48 3p12 213194_at ROBO1 0.150 495q32-q33.1 244686_at TCOF1 0.120 50 8q23.1 200638_s_at YWHAZ 0.26 0.230.012 51 10q23.31 205235_s_at MPHOSPH1 0.40 0.16 0.050 ^(†) Correlationbetween gene expression signal and the CKS1B amplification index (N =197, all patients with both GEP and FISH 1q21. Blank cells correspond toinsignificant correlations (nominal P > 0.05, no multiple comparisonsadjustment). ^(‡)Correlation between each gene's log-scale expressionand CKS1B log-scale expression. Rows with CKS1B |r| >= 0.4 are formattedbold. *Correlation between each gene's log-scale expression and thePCLI. ^(a)PH regression of overall survival on quartile 1 expression foreach gene, adjusted for FISH 13 80% cytogenetic abnormalities, B2M > 4,CRP > 4, ALB < 3.5 and PCLI (N = 277, 74 patients are missing at leastone measurement, 17 are missing FISH13, 4 are missing CA, 10 are missingone of B2M, CRP and ALB and 46 are missing PCLI: P = 0.51 for a log ranktest of the effect of exclusion due to missing measurements). TheseP-values are not adjusted for the quartile 1 log rank significancetesting that determined the ranks in column 1.

TABLE 6B Quartile 1 gene probe sets satisfying FDR 2.5% cutoff CKS1BAmplification Adjusted Rank Index CKS1B PCLI Survival (Q1) ChromosomeProbe set Symbol r ^(†) r^(‡) r* P-value^(a) 1 9q31.3 201921_at GNG10−0.20 −0.30 0.600 2 1p13 227278_at NA −0.12 0.900 3 Xp22.3 209740_s_atPNPLA4 0.029 4 20q11.21 227547_at NA −0.29 −0.28 −0.15 0.630 5 10q25.1225582_at KIAA1754 −0.21 −0.32 0.003 6 1p13.2 200850_s_at AHCYL1 −0.130.019 7 1p13.3 213628_at MCLC −0.30 −0.28 −0.15 0.440 8 1p22 209717_atEVI5 −0.33 −0.29 −0.16 0.870 9 1p13.3 222495_at AD-020 −0.30 −0.24 −0.200.920 10 6p21.31 1557277_a_at NA −0.11 0.460 11 1p22.1 1554736_at PARG1−0.20 −0.11 0.280 12 1p22 218924_s_at CTBS −0.16 −0.11 −0.13 0.460 139p13.2 226954_at NA −0.22 −0.40 0.090 14 1p34 202838_at FUCA1 −0.17−0.23 0.066 15 13q14 230192_at RFP2 −0.28 −0.18 0.880 16 12q13.1148106_at FLJ20489 −0.23 −0.23 −0.11 0.300 17 11q13.1 237964_at NA −0.16−0.20 0.044 18 2p22-p21 202729_s_at LTBP1 −0.24 −0.21 0.097 19 1p13.1212435_at NA −0.21 −0.21 −0.11 0.034 ^(†) Correlation between eachgene's log-scale expression and the CKS1B amplification index (N = 197,all patients with both GEP and FISH 1q21). Blank cells correspond toinsignificant correlations. ^(‡)Correlation between each gene'slog-scale expression and CKS1B log-scale expression. Rows with CKS1B|r| >s= 0.4 are formatted bold. *Correlation between each gene'slog-scale expression and the PCLI. ^(a)PH regression of overall survivalon Quartile 1 expression for each gene, adjusted for FISH 13 80%,cytogenetic abnormalities, B2M > 4, CRP > 4, ALB < 3.5 and PCLI (N =277, 74 patients are missing at least one measurement, 17 are missingFISH 13, 4 are missing CA, 10 are missing one of B2M, CRP and ALB, and46 are missing PCLI: P = 0.51 for a log rank test of the effect ofexclusion due to missing measurements). These P-values are not adjustedfor the quartile 1 log rank significance testing that determined theranks in column 1.

Although 10% of the genes on the microarray were derived from chromosome1, 30% of the retained genes were derived from chromosome 1, 30% of theretained genes were derived from this chromosome (P<0.0001) with 12 of51 quartile 4 genes (23.5%) mapped to chromosome 1q and 9 of 19 quartile1 genes (47%) mapped to chromosome 1p (Table 7).

TABLE 7 Chromosome Distribution of 2.5% FDR Probe Sets U133Plus2.0 Q1 Q4Combined No. of No. of No. of No. of No. of Chromosome Genes % Genes %Genes Chromosome Genes % Genes 1 3,659 9.9 9 47.4 12 1 3,659 9.9 9 22,522 6.9 1 5.3 2 2 2,522 6.9 1 3 2,116 5.8 0 0.0 7 3 2,116 5.8 0 41,456 4.0 0 0.0 0 4 1,456 4.0 0 5 1,718 4.7 0 0.0 2 5 1,718 4.7 0 62,005 5.4 1 5.3 1 6 2.005 5.4 1 7 1,798 4.9 0 0.0 2 7 1,798 4.9 0 81,311 3.6 0 0.0 4 8 1,311 3.6 0 9 1,463 4.0 2 10.5 0 9 1,463 4.0 2 101,444 3.9 1 5.3 2 10 1,444 3.9 1 11 2,069 5.6 1 5.3 1 11 2,069 5.6 1 121,927 5.2 1 5.3 3 12 1,927 5.2 1 13 730 2.0 1 5.3 0 13 730 2.0 1 141,195 3.2 0 0.0 0 14 1,195 3.2 0 15 1,152 3.1 0 0.0 0 15 1,152 3.1 0 161,507 4.1 0 0.0 2 16 1,507 4.1 0 17 2,115 5.7 0 0.0 3 17 2,115 5.7 0 18582 1.6 0 0.0 1 18 582 1.6 0 19 2,222 6.0 0 0.0 3 19 2,222 6.0 0 201,072 2.9 1 5.3 2 20 1,072 2.9 1 21 468 1.3 0 0.0 1 21 468 1.3 0 22 9062.5 0 0.0 1 22 906 2.5 0 X 1,273 3.5 1 5.3 2 X 1,273 3.5 1 Y 80 0.2 00.0 0 Y 80 0.2 0 m 5 0.0 0 0.0 0 m 5 0.0 0 36,795 19 51 36,795 19Unknown 17,880 Unknown 17,880 54,675 54,675 *An exact test for binomialproportions was used to compare the proportion of retained probe setsmapping to chromosome 1 to the proportion for the entire array.

The log-scale expression levels of proliferation-associated genes tendedto have high correlations with CKS1B (Table 6). In addition, 25 of 29(86%) genes, significantly correlated with the plasma cell labelingindex, were strongly correlated with CKS1B, suggesting that this geneparticipated in a proliferation signaling network in patients with highrisk disease. CKS1B is an independent predictor of overall survivalafter adjustment for chromosome 13 deletion, cytogenetic abnormalities,clinical prognostic factors and labeling index (P=0.007, Table 6, lastcolumn, row 17). Adjusted P-values are provided for other 69 genes forcomparison and it is evident that few other chromosome 1 genes are bothstrong independent predictors of survival, proliferation and CKS1B geneamplification.

Since amplification of 1q21 was associated with myeloma progression(Shaughnessy et al., 2000), the over-representation of 1q genes amongthe list of 70 justified a focus on this region in search for amolecular basis of high-risk myeloma; 4 genes (TAGNL2, PSMD4, MGC57827,CKS1B) map to 1q21, among which CKS1B quartile 4 membership was moststrongly associated with survival in unadjusted log rank tests (i.e.according to the list order of Table 6).

DNA synthesis is mediated by the action of the cyclin E/CDK2 complex,which is in turn negatively regulated by the cyclin-dependent kinaseinhibitor p27Kip1 (Sherr, et al., 1999). The small evolutionarilyconserved protein CKS1 is required for SCFSkp2-mediated ubiquitinylationand proteasomal degradation of cyclin-dependent kinase inhibitor p27Kip1(Ganoth, et al., 2001; Spruck, et al., 2001). p27Kip1 degradation notonly permits DNA replication but also ensures the correct progression ofcells through S phase into mitosis (Nakayama, et al., 2004) and Cksproteins interact with the proteasome to control the proteolysis ofmitotic cyclins by way of regulating the transcriptional activity ofCDC20 (Morris, et al., 2003), a regulatory subunit of theanaphase-promoting complex/cyclosome ubiquitin ligase (Peters, 2002).Thus, CKS1 and the SCFSkp2-p27Kip1-Cdk1/2 axis appear to be importantfor both DNA synthesis and mitosis (Pagano, 2004). The low p27Kip1protein levels in cancer cells, in the absence of gene mutations, hasprompted speculation that hyper-activation of CKS1B and/or SKP2, mayaccount for the low levels of p27Kip1 (Slingerland and Pagano, 2000).Given its well-documented role in regulating cell cycle progression, itsmap location, link to myeloma cell proliferation and patient survival,CKS1B was considered a candidate gene, the inappropriate expression ofwhich may promote an aggressive phenotype.

As was true for all the gene transcripts listed in Table 6, CKS1B levelswere strongly correlated with clinical outcome (FIGS. 6A-B): 25 deathshad occurred among 88 patients with quartile 4 expression levelscompared to only 39 among the 263 patients with quartile 1-3 levels(p<0.0001, false discovery rate, 2.5%); this was also true forevent-free survival (34 of 88 in the quartile 4 cohort had experiencedan event compared to 64 of 263 in the remainder; p<0.0001). Levels ofSKP2, the CKS1B partner gene, were not significantly associated withsurvival (P=0.3).

Next, whether CKS1B gene expression was linked to gene copy number wasexamined Up to 8 copies of CKS1B were detected in metaphases of primarysamples (FIG. 7A). Interphase fluorescence in situ hybridizationanalysis revealed 3 or more copies of CKS1B in 46% among 197 cases withconcurrent gene expression data. Expression levels were significantlylinked to CKS1B copy number (FIG. 7B). Conversely, amplificationincreased in frequency as CKS1B expression levels increased fromquartile 1 to quartile 4 (P<0.0001, Table 8).

TABLE 8 Relationship between CKS1B gene expression quartiles and CKS1Bamplification in newly diagnosed myeloma CKS1B Expression^(†) #AMPLIFIED % AMPLIFIED quartile 1^(‡) 9 20% n = 44 quartile 2 12 28% n =43 quartile 3 26 51% n = 51 quartile 4 44 75% n = 59 total 91 46% 197^(†)P < 0.0001. Amplification is defined as >=20% of cells with 3 or >=4CKS1B signals, for validation in conjunction with FIGS. 7c-d, asdescribed in the Methods. Other tables use the CKS1B amplification indexand its optimal cutoff. ^(‡)Quartile assignments based upon 351 patientswith GE

Examination of CKS1B gene amplification in the context of expressionlevels of the 70 genes (Table 6) revealed, as expected, correlationswith genes on chromosome 1q21 but, importantly, also with genes linkedto cell proliferation not mapping to 1q21. The BAC clone used toevaluate CKS1B gene copy number also measured the copy number of PBXIP1(mapping centromeric to CKS1B) and PB591, LENEP, ZFP67, FLJ32934, ADAM15and EFNA4 (all mapping telomeric to CKS1B). In examining therelationship between gene copy number and the expression levels of thesegenes (Table 9), RNA expression was most strongly correlated with DNAcopy number in the case of CKS1B. Importantly, none of the other genesmapping to this BAC were among the 70 linked to short survival.

TABLE 9 Relationship of quartile 4 gene expression to amplification forgenes located on bacterial artificial chromosome (BAC) used to measure1q21 amplification. Not Amplified* Amplifi- Log Amplified(Amplification. cation Rank n/ Index. >= 46) P- Index N/ Symbol 129 (%)n/68 (%) Value^(†) Symbol 129 PBXIP1 24 (18.6) 28 (41.2) 0.0012 PBXIP124 CKS1B 20 (15.5) 39 (57.4) <0.0001 CKS1B 20 PB591 23 (17.8) 38 (55.9)<0.0001 PB591 23 LENEP 31 (24.0) 18 (26.5) 0.8389 LENEP 31 ZFP67 27(20.9) 29 (42.6) 0.0023 ZFP67 27 FLJ32934 28 (21.7) 11 (16.2) 0.4606FLJ32934 28 ADAM15 23 (17.8) 29 (42.6) 0.0003 ADAM15 23 EFNA4 26 (20.2)23 (33.8) 0.0528 EFNA4 26 *The 0-100 scale CKS1B amplification index isa weighted sum of the proportions of clonal cells with 3 copies of CKS1Band >=4 copies of CKS1B, defined by (.34 * %3 copies + .66 * % >= 4copies)/.66 ^(†)For a test of the independence of amplification and 4thquartile membership (N = 197) ^(‡)Correlation between each gene'sexpression and the 0-100 scale CKS1B amplification index ^(a)Log ranktest for association of Q4 membership and overall survival (N = 351, 64deaths)

Further, the association of CKS1B over-expression with survival andevent-free survival was validated in a cohort of 224 patients lackingmicroarray data. CKS1B amplification levels were inversely correlatedwith both event-free survival (P<0.0001) and overall survival (P<0.0001,FIG. 7C). These effects were also observed when all 421 patients wereconsidered (overall survival, P<0.0001, FIG. 7D; event-free survival,P<0.0001).

Multivariate proportional hazards analyses were performed using the 369patients with both CKS1B amplification samples and complete risk factordata (Table 10). The 3 genetic risk factors (CKS1B amplification,chromosome 13q14 deletion, metaphase karyotype abnormalities) allindependently conferred both inferior event-free and overall survival,whereas hypoalbuminemia was the only one of three standard prognosticfactors that retained adverse implications for both endpoints examinedCollectively, these 6 variables accounted for 46% and 33% of variabilityin survival and event-free survival, respectively, with the 3 standard,non-genetic parameters contributing only an additional 7.2% and 7.4%.CKS1B amplification was an independent predictor of outcome both as a0-100 scale index and a two-group category (Tables 10A and B), afteradjustments for the variables mentioned above and for the plasma celllabeling index.

TABLE 10A Multivariate proportional hazards analysis^(†) (n = 369)Event-Free Survival Survival % HR P Cumulative r² HR P Cumulative r²CKS1B Amplification 1.009 0.002 0.160 1.011 0.002 0.219 Index (0-100)FISH Chromosome 25.5 1.786 0.006 0.224 1.879 0.014 0.308 13 DeletionAbnormal Karyotype 35.0 1.875 0.001 0.272 2.298 <0.001 0.393Beta-2-microglobulin >= 35.8 1.478 0.046 0.305 1.396 0.170 0.422 4 mg/LC-reactive protein >= 63.4 1.533 0.028 0.320 1.586 0.055 0.448 4 mg/LAlbumin < 3.5 g/dL 16.5 1.660 0.019 0.336 1.698 0.044 0.461Events/Deaths 127 84

TABLE 10B Multivariate proportional hazards analysis^(†) (n = 369)Event-Free Survival Survival % HR P Cumulative r² HR P Cumulative r²CKS1B Amplification 32.5 1.68 0.008 0.132 2.12 0.001 0.207 Index >= 46FISH Chromosome 25.5 1.74 0.010 0.204 1.83 0.020 0.293 13 DeletionAbnormal Karyotype 35.0 1.94 <0.001 0.257 2.33 <0.001 0.383Beta-2-microglobulin >= 35.8 1.52 0.033 0.293 1.43 0.140 0.417 4 mg/LC-reactive protein >= 63.4 1.49 0.038 0.312 1.56 0.060 0.443 4 mg/LAlbumin < 3.5 g/dL 16.5 1.69 0.016 0.331 1.73 0.035 0.455 Events/Deaths127 84

Paired CKS1B expression data at diagnosis and relapse, available in 32cases, revealed increased expression in 84% at relapse (P=0.0001, FIG.8), which was especially prominent in patients with quartile 1-3expression levels at diagnosis. Paired CKS1B copy number data atdiagnosis and relapse were available in 17 patients: of 7 lackingamplification at diagnosis, 4 acquired>=3 copies at relapse; of 10 caseswith 3 copies at diagnosis, 4 had acquired>=4 copies at relapse but 2cases with 4 or more copies at diagnosis exhibited no furtheramplification at relapse. These data suggested that CKS1Bamplification/over-expression was also associated with diseaseprogression.

The frequency of CKS1B quartile 4 expression varied among previouslyreported genetic subgroups (Table 11). With respect to geneexpression-based translocations, nearly two-thirds of patients with MAFor MAFB activation, one-third each with FGFR3/MMSET and CCND1activation, and only 18% without translocations had CKS1Bhyper-activation (P<0.0001). When examined in the context of metaphasekaryotypes, CKS1B quartile 4 expression was present in approximately 20%of cases with hyperdiploid or normal, i.e. uninformative, karyotypes,whereas this feature was seen in nearly 50% of patients with hypodiploidand other cytogenetic abnormalities (P=0.0002).

In a separate multivariate analysis that adjusted for genetic subgroups,CKS1B quartile 4 expression remained an independent adverse outcomepredictor (Table 11); the gene expression-derived translocation categoryas a whole conferred inferior event-free (P=0.034) but not of overallsurvival (P=0.261); however, consistent with Fonseca et al., 2004),CCND1 activation impacted both endpoints favorably. While not adjustedfor the multiple log rank tests that identified the 70 genes, thisanalysis, suggests that CKS1B expression retains explanatory powerwithin relevant genetic subgroups.

TABLE 11A Relationship between genetic abnormalities and CKS1Bexpression quartiles CKS1B Abnormality Q4 Category^(†) n/347 (%) n (%)P-Value* Expression-derived translocation t(11; 14) 60 (17.3) 20 (33.3)<0.0001 t(4; 14) 48 (13.8) 17 (35.4) t(14; 16) & t(14; 20) 14 (4.0)  9(64.3) No Translocation Spike 225 (64.8)  41 (18.2) Metaphase karyotypeHyperdiploid 55 (15.9) 10 (18.2) 0.0002 Non-hyperdiploid 48 (13.8) 24(50.0) Other Cytogenetics Abnormality 9 (2.6) 4 (44.4) No CytogeneticsAbnormality 235 (67.7)  49 (20.9) ^(†)Translocations were determinedfrom the expression spikes t(11; 14) = CCND1, t(4:14) = FGFR3/MMSET,t(14; 16) = MAF and t(14; 20) = MAFB. Aneuploidy and other cytogeneticabnormalities were determined from cytogenetics, for which 4observations were missing. *Fisher's exact test of the independence ofeach category and CKS1B 4th quartile membership. Under the nullhypothesis, Q4 contains on average 25% of patients within each level,corresponding to a proportional distribution across Q1-3 and Q4.

TABLE 11B Multivariate analysis of CKS1B quartile 4 expression andcytogenetic abnormalities^(†) Event-Free Survival Survival HR P HR PCKS1B Q4 1.97 0.003 2.16 0.005 Expression-derived translocation t(11;14) 0.59 0.034 0.82 0.261 t(4; 14) 1.67 1.77 t(14; 16) & t(14; 20) 1.481.12 Metaphase karyotype Hyperdiploid 1.75 0.006 1.84 0.013Non-hyperdiploid 2.29 2.56 Other Cytogenetics Abnormality 2.35 2.71 r²0.218 0.223 Events/Deaths 97 63 ^(†)N = 347. Of 351 patients withexpression data, 4 are missing cytogenetics. ^(‡)Partial likelihoodratio test for the overall effect of the category.

Additionally, Western blot analysis of nuclear protein from plasma cellsfrom 27 newly diagnosed myeloma cases and 7 myeloma cell lines showed astrong correlation between CKS1B mRNA and protein, but no correlationbetween mRNA and protein levels for p27^(Kip1). However, CKS1B proteinand p27^(Kip1) protein levels showed an inverse correlation (FIG. 8).Cytoplasmic and non-phosphorylated-thr-187-p27^(Kip1) levels were notaltered in myeloma cell lysates with respect to CKS1B expression (datanot shown). Levels of p27^(Kip1) protein were not correlated with themRNA levels of SKP2 (data not shown).

Example 11 The Effect of Bortezomib on Patients with Abnormal CopyNumbers of Genes Located in Chromosomes 1 and 13

Many recurrent genomic aberrations in MM have been discovered during thepast two decades. In particular, the deletion of chromosome 13 and theamplification of 1q21 are associated with poor diagnosis of patient withmultiple myeloma. The following example pertain to clinical resultsobtained from newly diagnosed multiple myeloma patients.

The total therapy 2 protocol enrolled 668 TT2 patients and the totaltherapy 3 protocols (TT3 and TT3b) enrolled 480 patients. A keydifference between TT2 and TT3 is the administration of bortezomib inTT3 protocols. Bone marrow specimens were obtained at baseline, aftertreatment, and at relapse. FISH analysis has been carried out usingprobes for chromosome 1q21 (CKS1B), 1p13 (AHCYL1), 13q14 (D13S31/RB1)and 13qtel (D13S285) (FIG. 10). By simultaneously staining the tumorcells with the probes listed above labeled with nucleotides conjugatedwith red and green fluorophores and an AMCA-labeled antibody thatrecognizes kappa or lambda light chain producing PC, clonally restrictedtumor cells can be distinguished from a heterogeneous population of bonemarrow cells under fluorescence microscopy. This technique was termedTRI-FISH for Triple color-FISH (FIG. 11). At least 100 light chainrestricted cells are scored from each sample. Of 480 TT3/TT3b patients,247 baseline specimens have been analyzed by FISH with the 4 probes. Inaddition, progress has been made to reexamine the specimens from TT2patients with the 4 probes and 165 of these baseline specimens have beenanalyzed to date.

A summary of FISH results on baseline specimens from the combined TT2and TT3/TT3b are summarized in Table 12

TABLE 12 FISH on the Baseline Myeloma Specimens (sample size: 517) 1q211p13 D13S31 D13S285 momosomy 3% 21% 45% 42% disomy 60% 74% 52% 54%trisomy 26% 3% 1% 3% tetrasomy or more 12% 2% 1% 1%

The prognostic significance of FISH-defined copy number abnormalitiesfor 1q21, 1p13 and 13q14 alone or in combination was investigated.Kaplan-Meier (KM) analysis of overall survival (FIGS. 12A-D, 14A-D,16A-D, 22A-C, 23A-C, 24A-C, 25A-C, 26A, 27A, 28A and 29A) and event freesurvival (FIGS. 13A-D, 15A-D, 17A-D, 22D-F, 23D-F, 24D-F, 25D-F, 26B,27B, 28B and 29B) related to FISH abnormalities of 1q21 (FIGS. 12A-D,13A-D, 22A-F, 23A-F, 24A-F, 28A-B and 29A-B) 1p13 (FIGS. 14A-D, 15A-D,24A-F and 26A-B) and 13q14 (FIGS. 16A-D, 17A-D, 25A-F and 27A-B) in theentire test cohort (upper left panel), TT3/TT3b (upper right panel),TT2-thalidomide (lower left panel) and TT2+thalidomide (lower rightpanel). KM analysis of overall survival and event free survival based oninteractions between gains of 1q21 and loss of 1p13 (FIGS. 18A-D and19A-D) and gain of 1q21 and deletion of 13q14 (FIGS. 20A-D and 21A-D)are shown as well. Univariate and multivariate analyses for these FISHabnormalities and molecular variables, standard prognostic variables andrandomization to thalidomide are presented for TT2 (Tables 13a-d) andTT3/TT3b (Tables 14a-d).

TABLE 13a Univariate and multivariate associations of FISH-definedGain/Amp1q21 (3 or 4 copies) and molecular and clinical variables withoverall and event-free survival in TT2 Univariate Overall Survival inTT2 Event-free survival TT2 Variable n/N (%) HR (95% CI) P-value HR (95%CI) P-value Age >= 65 yr 25/125 (20%) 1.47 (0.76, 2.81) 0.251 1.27(0.76, 2.11) 0.359 Albumin < 3.5 g/dL 18/125 (14%) 1.33 (0.62, 2.83)0.465 1.28 (0.72, 2.26) 0.404 B2M >= 3.5 mg/L 47/125 (38%) 2.12 (1.21,3.70) 0.008 1.46 (0.96, 2.22) 0.079 B2M > 5.5 mg/L 27/125 (22%) 2.13(1.17, 3.86) 0.013 1.40 (0.86, 2.26) 0.174 Creatinine >= 2 mg/dL 15/122(12%) 1.90 (0.92, 3.92) 0.083 1.54 (0.87, 2.73) 0.138 Hb < 10 g/dL36/125 (29%) 1.10 (0.61, 1.99) 0.756 0.98 (0.63, 1.54) 0.939 LDH >= 190U/L 33/125 (26%) 2.17 (1.22, 3.85) 0.008 1.42 (0.90, 2.25) 0.132 CRP >=8 mg/L 53/123 (43%) 1.16 (0.66, 2.02) 0.614 0.74 (0.48, 1.13) 0.163Cytogenetic Abnormalities 35/124 (28%) 1.71 (0.95, 3.08) 0.074 1.67(1.07, 2.61) 0.024 GEP High Risk 13/82 (16%)  9.11 (4.22, 19.67) <.0013.51 (1.78, 6.89) <.001 Randomization to Thalidomide 61/125 (49%) 0.94(0.54, 1.65) 0.841 0.78 (0.52, 1.19) 0.252 Gain/Amp1q21 53/125 (42%)1.58 (0.91, 2.75) 0.108 1.57 (1.03, 2.37) 0.034 Testing theRandomization to Thalidomide 61/125 (49%) 1.10 (0.49, 2.45) 0.812 1.08(0.61, 1.92) 0.785 interaction Gain/Amp1q21 53/125 (42%) 2.11 (0.99,4.52) 0.054 3.26 (1.83, 5.82) <.001 between +Thal/Gain/Amp1q21 32/125(26%) 0.58 (0.19, 1.79) 0.346 0.31 (0.13, 0.72) 0.006 Thalidomide GEPIFN >= 11 15/82 (18%) 1.19 (0.49, 2.88) 0.703 0.92 (0.47, 1.82) 0.812and FISH GEP NFKB score >= 11 25/82 (30%) 0.58 (0.25, 1.34) 0.201 1.04(0.60, 1.81) 0.882 GEP Centrosome Index >= 3 13/82 (16%) 2.35 (1.06,5.21) 0.035 1.86 (0.98, 3.52) 0.057 GEP Proliferation Index >= 10 9/82(11%) 2.95 (1.27, 6.83) 0.012 2.05 (0.96, 4.39) 0.063 GEP Poly PCScore >= 13 1/82 (1%)  2.83 (0.38, 20.97) 0.309  1.45 (0.20, 10.55)0.715 Multivariate Overall Survival in TT2 Event-free survival TT2Variable n/N (%) HR (95% CI) P-value HR (95% CI) P-value Randomizationto Thalidomide 37/78 (47%) 2.32 (0.74, 7.34) 0.151 1.37 (0.63, 2.95)0.429 Gain/Amp1q21 37/78 (47%) 2.08 (0.71, 6.13) 0.184 2.51 (1.19, 5.30)0.016 +Thal/Gain/Amp1q21 22/78 (28%) 0.24 (0.06, 1.06) 0.059 0.36 (0.13,1.04) 0.059 B2M >= 3.5 mg/L 31/78 (40%) 2.29 (1.03, 5.11) 0.042 NS2 NS2LDH >= 190 U/L 23/78 (29%) 2.61 (1.17, 5.81) 0.019 1.84 (1.05, 3.21)0.033 GEP High Risk 12/78 (15%) 12.61 (4.69, 33.90) <.001 3.09 (1.46,6.56) 0.003 GEP Poly PC Score >= 13 1/78 (1%)  15.37 (1.67, 141.14)0.016 NS2 NS2 HR—Hazard Ratio, 95% CI—95% Confidence Interval, P-valuefrom Wald Chi-Square Test in Cox Regression

TABLE 13b Univariate and multivariate associations of FISH-definedAmp1q21 (4 copies) and molecular and clinical variables with overall andevent-free survival in TT2 Overall Survival in TT2 Event-free survivalTT2 Variable n/N (%) HR (95% CI) P-value HR (95% CI) P-value UnivariateAge >= 65 yr 20/89 (22%) 2.05 (0.96, 4.37) 0.062 1.31 (0.73, 2.35) 0.361Albumin < 3.5 g/dL 9/89 (10%) 0.92 (0.28, 3.04) 0.897 1.67 (0.79, 3.51)0.179 B2M >= 3.5 mg/L 32/89 (36%) 1.88 (0.94, 3.78) 0.075 1.28 (0.77,2.13) 0.341 B2M > 5.5 mg/L 16/89 (18%) 1.54 (0.69, 3.44) 0.288 1.03(0.55, 1.93) 0.929 Creatinine >= 2 mg/dL 8/86 (9%) 1.37 (0.48, 3.93)0.556 1.05 (0.48, 2.31) 0.904 Hb < 10 g/dL 22/89 (25%) 0.89 (0.40, 1.98)0.776 0.92 (0.52, 1.62) 0.774 LDH >= 190 U/L 21/89 (24%) 1.75 (0.83,3.70) 0.145 1.29 (0.72, 2.31) 0.397 CRP >= 8 mg/L 38/89 (43%) 0.96(0.48, 1.93) 0.913 0.67 (0.40, 1.11) 0.124 Cytogenetic Abnormalities22/88 (25%) 1.24 (0.55, 2.77) 0.607 1.85 (1.07, 3.18) 0.028 GEP HighRisk 4/55 (7%) 18.90 (4.82, 74.14) <.001  6.12 (1.94, 19.35) 0.002Randomization Thalidomide 39/89 (44%) 1.10 (0.55, 2.22) 0.781 0.97(0.59, 1.60) 0.899 Amp1q21 17/89 (19%) 1.42 (0.61, 3.29) 0.416 1.60(0.88, 2.91) 0.123 Testing the Randomization Thalidomide 39/89 (44%)1.11 (0.50, 2.48) 0.795 1.08 (0.61, 1.92) 0.785 interaction Amp1q2117/89 (19%) 1.61 (0.47, 5.59) 0.451 2.91 (1.25, 6.78) 0.013 between+Thal/Amp1q21 10/89 (11%) 0.77 (0.14, 4.24) 0.769 0.37 (0.11, 1.23)0.105 Thalidomide GEP IFN >= 11 13/55 (24%) 1.06 (0.35, 3.24) 0.913 0.88(0.40, 1.92) 0.741 and FISH GEP NFKB score >= 11 20/55 (36%) 0.50 (0.16,1.51) 0.217 0.92 (0.47, 1.81) 0.815 GEP Centrosome Index >= 3 6/55 (11%)1.25 (0.29, 5.45) 0.766 1.55 (0.60, 4.00) 0.361 GEP ProliferationIndex >= 10 1/55 (2%) 5.95 (0.74, 47.65) 0.093  2.84 (0.38, 21.46) 0.311GEP Poly PC Score >= 13 1/55 (2%) 3.98 (0.51, 30.86) 0.187  1.87 (0.25,13.92) 0.539 Multivariate Randomization to Thalidomide 21/52 (40%) 2.24(0.70, 7.11) 0.173 2.23 (0.94, 5.31) 0.070 Amp1q21 11/52 (21%) 1.05(0.17, 6.27) 0.961 2.10 (0.73, 6.01) 0.168 +Thal/Amp1q21 6/52 (12%) 1.14(0.09, 13.63) 0.920 0.30 (0.06, 1.38) 0.122 Cytogenetic Abnormalities14/52 (27%) NS2 NS2 3.07 (1.33, 7.10) 0.009 LDH >= 190 U/L 13/52 (25%)3.95 (1.34, 11.61) 0.013 NS2 NS2 GEP High Risk 4/52 (8%) 37.16 (6.52,211.73) <.001 NS2 NS2 GEP Poly PC Score >= 13 1/52 (2%) 15.66 (1.50,163.66) 0.022 NS2 NS2 HR—Hazard Ratio, 95% CI—95% Confidence Interval,P-value from Wald Chi-Square Test in Cox Regression NS2—Multivariateresults not statistically significant at 0.05 level. All univariatep-values reported regardless of significance. Multivariate model usesstepwise selection with entry level 0.1 and variable remains if meetsthe 0.05 level. A multivariate p-value greater than 0.05 indicatesvariable forced into model with significant variables chosen usingstepwise selection.

TABLE 13c Univariate and multivariate associations of FISH-defineddel1p13 and molecular and clinical variables with overall and event-freesurvival in TT2 Overall Survival in TT2 Event-free survival TT2 Variablen/N (%) HR (95% CI) P-value HR (95% CI) P-value Univariate Age >= 65 yr25/123 (20%) 1.38 (0.72, 2.65) 0.334 1.21 (0.73, 2.02) 0.458 Albumin <3.5 g/dL 17/122 (14%) 1.41 (0.66, 3.01) 0.369 1.49 (0.84, 2.64) 0.174B2M >= 3.5 mg/L 47/123 (38%) 1.88 (1.07, 3.28) 0.027 1.38 (0.90, 2.11)0.138 B2M > 5.5 mg/L 26/123 (21%) 2.00 (1.09, 3.67) 0.025 1.39 (0.85,2.28) 0.184 Creatinine >= 2 mg/dL 14/120 (12%) 1.81 (0.85, 3.86) 0.1271.60 (0.89, 2.89) 0.120 Hb < 10 g/dL 35/123 (28%) 1.12 (0.62, 2.02)0.718 0.96 (0.61, 1.52) 0.869 LDH >= 190 U/L 34/123 (28%) 1.98 (1.12,3.52) 0.020 1.32 (0.83, 2.09) 0.238 CRP >= 8 mg/L 51/121 (42%) 1.08(0.62, 1.89) 0.787 0.70 (0.46, 1.08) 0.109 Cytogenetic Abnormalities35/122 (29%) 1.63 (0.91, 2.94) 0.103 1.61 (1.03, 2.51) 0.038 GEP HighRisk 13/82 (16%) 8.62 (4.04, 18.41) <.001 3.32 (1.69, 6.51) <.001Randomization to Thalidomide 56/123 (46%) 1.04 (0.60, 1.82) 0.888 0.83(0.54, 1.27) 0.388 del1p13 24/123 (20%) 1.85 (0.97, 3.56) 0.063 1.52(0.90, 2.55) 0.116 Testing the Randomization to Thalidomide 56/123 (46%)0.91 (0.48, 1.73) 0.772 0.75 (0.47, 1.21) 0.237 interaction del1p1324/123 (20%) 1.36 (0.55, 3.35) 0.507 1.16 (0.58, 2.31) 0.679 between+Thal/del1p13 11/123 (9%) 2.11 (0.57, 7.78) 0.263 1.91 (0.67, 5.45)0.224 Thalidomide GEP IFN >= 11 15/82 (18%) 1.12 (0.46, 2.69) 0.809 0.90(0.46, 1.78) 0.766 and FISH GEP NFKB score >= 11 26/82 (32%) 0.62 (0.28,1.36) 0.232 1.04 (0.60, 1.79) 0.887 GEP Centrosome Index >= 3 12/82(15%) 2.68 (1.21, 5.93) 0.015 1.84 (0.96, 3.56) 0.068 GEP ProliferationIndex >= 10 9/82 (11%) 2.79 (1.21, 6.44) 0.016 1.99 (0.93, 4.24) 0.076GEP Poly PC Score >= 13 1/82 (1%) 2.66 (0.36, 19.72) 0.337 1.35 (0.19,9.80) 0.768 Multivariate Randomization to Thalidomide 35/77 (45%) 1.15(0.49, 2.70) 0.746 0.84 (0.45, 1.57) 0.589 del1p13 19/77 (25%) 1.77(0.62, 5.10) 0.288 1.03 (0.45, 2.39) 0.941 +Thal/del1p13 10/77 (13%)0.51 (0.11, 2.47) 0.404 1.57 (0.41, 5.99) 0.510 GEP High Risk 12/77(16%) 10.24 (3.86, 27.13) <.001 2.78 (1.15, 6.72) 0.023 LDH >= 190 U/L24/77 (31%) 2.69 (1.31, 5.53) 0.007 NS2 NS2 HR—Hazard Ratio, 95% CI—95%Confidence Interval, P-value from Wald Chi-Square Test in Cox RegressionNS2—Multivariate results not statistically significant at 0.05 level.All univariate p-values reported regardless of significance.Multivariate model uses stepwise selection with entry level 0.1 andvariable remains if meets the 0.05 level. A multivariate p-value greaterthan 0.05 indicates variable forced into model with significantvariables chosen using stepwise selection.

TABLE 13d Univariate and multivariate associations of FISH defineddel13q14 and molecular and clinical variables with overall andevent-free survival in TT2 Overall Survival in TT2 Event-free survivalTT2 Variable n/N (%) HR (95% CI) P-value HR (95% CI) P-value UnivariateAge >= 65 yr 25/126 (20%) 1.42 (0.74, 2.72) 0.293 1.23 (0.74, 2.05)0.416 Albumin < 3.5 g/dL 17/125 (14%) 1.44 (0.67, 3.05) 0.348 1.48(0.84, 2.62) 0.177 B2M >= 3.5 mg/L 48/126 (38%) 1.97 (1.13, 3.42) 0.0161.37 (0.90, 2.08) 0.144 B2M > 5.5 mg/L 27/126 (21%) 2.06 (1.14, 3.73)0.017 1.36 (0.84, 2.20) 0.208 Creatinine >= 2 mg/dL 15/123 (12%) 1.85(0.90, 3.82) 0.095 1.51 (0.85, 2.67) 0.160 Hb < 10 g/dL 35/126 (28%)1.13 (0.63, 2.05) 0.677 0.97 (0.61, 1.52) 0.885 LDH >= 190 U/L 34/126(27%) 2.01 (1.14, 3.57) 0.016 1.33 (0.84, 2.10) 0.224 CRP >= 8 mg/L53/124 (43%) 1.10 (0.63, 1.91) 0.739 0.71 (0.47, 1.09) 0.119 CytogeneticAbnormalities 35/125 (28%) 1.66 (0.92, 2.98) 0.091 1.62 (1.04, 2.53)0.033 GEP High Risk 13/83 (16%) 8.78 (4.11, 18.76) <.001 3.33 (1.70,6.53) <.001 Randomization to Thalidomide 59/126 (47%) 1.00 (0.58, 1.74)0.987 0.82 (0.54, 1.24) 0.343 del13q14 75/126 (60%) 1.08 (0.62, 1.90)0.775 1.07 (0.70, 1.61) 0.763 Testing the Randomization to Thalidomide59/126 (47%) 1.19 (0.49, 2.88) 0.694 0.99 (0.52, 1.92) 0.986 interactiondel13q14 75/126 (60%) 1.24 (0.59, 2.60) 0.573 1.30 (0.75, 2.23) 0.353between +Thal/del13q14 40/126 (32%) 0.73 (0.24, 2.28) 0.593 0.70 (0.30,1.63) 0.403 Thalidomide GEP IFN >= 11 15/83 (18%) 1.14 (0.47, 2.75)0.772 0.89 (0.45, 1.77) 0.748 and FISH GEP NFKB score >= 11 26/83 (31%)0.63 (0.29, 1.40) 0.258 1.04 (0.60, 1.78) 0.893 GEP Centrosome Index >=3 13/83 (16%) 2.27 (1.03, 5.00) 0.043 1.79 (0.95, 3.38) 0.073 GEPProliferation Index >= 10 9/83 (11%) 2.84 (1.23, 6.56) 0.014 1.99 (0.93,4.24) 0.076 GEP Poly PC Score >= 13 1/83 (1%) 2.71 (0.37, 20.04) 0.3291.37 (0.19, 9.98) 0.754 Multivariate Randomization to Thalidomide 36/78(46%) 1.82 (0.55, 6.01) 0.326 1.19 (0.49, 2.87) 0.704 del13q14 51/78(65%) 1.19 (0.43, 3.30) 0.739 1.02 (0.50, 2.08) 0.958 +Thal/del13q1426/78 (33%) 0.39 (0.09, 1.73) 0.214 0.70 (0.23, 2.13) 0.534 GEP HighRisk 12/78 (15%) 12.09 (4.79, 30.54) <.001 3.73 (1.75, 7.94) <.001LDH >= 190 U/L 24/78 (31%) 2.82 (1.36, 5.81) 0.005 NS2 NS2 HR—HazardRatio, 95% CI—95% Confidence Interval, P-value from Wald Chi-Square Testin Cox Regression NS2—Multivariate results not statistically significantat 0.05 level. All univariate p-values reported regardless ofsignificance. Multivariate model uses stepwise selection with entrylevel 0.1 and variable remains if meets the 0.05 level. A multivariatep-value greater than 0.05 indicates variable forced into model withsignificant variables chosen using stepwise selection.

TABLE 14a Univariate and multivariate associations of FISH-definedGain/Amp1q21 (3 or 4 copies) and molecular and clinical variables withoverall and event-free survival in TT3 Overall Survival in TT3Event-free survival TT3 Variable n/N (%) HR (95% CI) P-value HR (95% CI)P-value Univariate Age >= 65 yr 68/240 (28%) 1.22 (0.64, 2.35) 0.5431.26 (0.71, 2.24) 0.425 Albumin < 3.5 g/dL 80/239 (33%) 1.90 (1.04,3.49) 0.038 1.77 (1.04, 3.02) 0.037 B2M >= 3.5 mg/L 120/238 (50%) 2.27(1.21, 4.27) 0.011 2.25 (1.29, 3.94) 0.004 B2M > 5.5 mg/L 62/238 (26%)3.95 (2.15, 7.25) <.001 4.18 (2.45, 7.15) <.001 Creatinine >= 2 mg/dL17/239 (7%) 2.09 (0.82, 5.32) 0.121 2.33 (1.05, 5.15) 0.037 Hb < 10 g/dL80/239 (33%) 1.74 (0.96, 3.18) 0.069 1.98 (1.17, 3.36) 0.011 LDH >= 190U/L 66/239 (28%) 2.17 (1.18, 3.98) 0.012 2.54 (1.49, 4.32) <.001 CRP >=8 mg/L 76/238 (32%) 1.39 (0.75, 2.59) 0.297 1.22 (0.70, 2.14) 0.477Cytogenetic Abnormalities 102/236 (43%) 3.03 (1.59, 5.75) <.001 2.39(1.38, 4.14) 0.002 GEP High Risk 43/227 (19%) 4.28 (2.30, 7.97) <.0014.23 (2.44, 7.33) <.001 Gain/Amp1q21 87/240 (36%) 1.60 (0.88, 2.91)0.126 1.58 (0.93, 2.68) 0.094 GEP IFN >= 11 55/227 (24%) 1.92 (1.02,3.61) 0.044 1.76 (1.00, 3.12) 0.050 GEP NFKB score >= 11 101/227 (44%)0.89 (0.48, 1.64) 0.705 0.90 (0.52, 1.55) 0.699 GEP Centrosome Index >=3 86/227 (38%) 3.89 (2.05, 7.37) <.001 2.84 (1.63, 4.93) <.001 GEPProliferation Index >= 10 28/227 (12%) 3.99 (2.09, 7.60) <.001 3.48(1.93, 6.26) <.001 GEP Poly PC Score >= 13 8/227 (4%) 1.31 (0.32, 5.45)0.707 1.01 (0.24, 4.15) 0.992 Multivariate Gain/Amp1q21 82/220 (37%)1.36 (0.69, 2.71) 0.378 1.27 (0.71, 2.27) 0.424 B2M > 5.5 mg/L 59/220(27%) NS2 NS2 2.50 (1.37, 4.58) 0.003 Creatinine >= 2 mg/dL 16/220 (7%)3.16 (1.17, 8.52) 0.023 NS2 NS2 Cytogenetic Abnormalities 96/220 (44%)2.83 (1.38, 5.78) 0.004 NS2 NS2 GEP High Risk 42/220 (19%) NS2 NS2 3.02(1.60, 5.69) <.001 GEP IFN >= 11 52/220 (24%) 3.20 (1.53, 6.68) 0.0022.23 (1.22, 4.07) 0.010 GEP Centrosome Index >= 3 83/220 (38%) 3.86(1.93, 7.74) <.001 NS2 NS2 GEP Poly PC Score >= 13 7/220 (3%)  7.40(1.49, 36.85) 0.015 NS2 NS2 HR—Hazard Ratio, 95% CI—95% ConfidenceInterval, P-value from Wald Chi-Square Test in Cox RegressionNS2—Multivariate results not statistically significant at 0.05 level.All univariate p-values reported regardless of significance.Multivariate model uses stepwise selection with entry level 0.1 andvariable remains if meets the 0.05 level. A multivariate p-value greaterthan 0.05 indicates variable forced into model with significantvariables chosen using stepwise selection.

TABLE 14b Univariate and multivariate associations of FISH-definedAmp1q21 (4 copies) and molecular and clinical variables with overall andevent-free survival in TT3 Overall Survival in TT3 Event-free survivalTT3 Variable n/N (%) HR (95% CI) P-value HR (95% CI) P-value UnivariateAge >= 65 yr 68/240 (28%) 1.22 (0.64, 2.35) 0.543 1.26 (0.71, 2.24)0.425 Albumin < 3.5 g/dL 80/239 (33%) 1.90 (1.04, 3.49) 0.038 1.77(1.04, 3.02) 0.037 B2M >= 3.5 mg/L 120/238 (50%) 2.27 (1.21, 4.27) 0.0112.25 (1.29, 3.94) 0.004 B2M > 5.5 mg/L 62/238 (26%) 3.95 (2.15, 7.25)<.001 4.18 (2.45, 7.15) <.001 Creatinine >= 2 mg/dL 17/239 (7%) 2.09(0.82, 5.32) 0.121 2.33 (1.05, 5.15) 0.037 Hb < 10 g/dL 80/239 (33%)1.74 (0.96, 3.18) 0.069 1.98 (1.17, 3.36) 0.011 LDH >= 190 U/L 66/239(28%) 2.17 (1.18, 3.98) 0.012 2.54 (1.49, 4.32) <.001 CRP >= 8 mg/L76/238 (32%) 1.39 (0.75, 2.59) 0.297 1.22 (0.70, 2.14) 0.477 CytogeneticAbnormalities 102/236 (43%) 3.03 (1.59, 5.75) <.001 2.39 (1.38, 4.14)0.002 GEP High Risk 43/227 (19%) 4.28 (2.30, 7.97) <.001 4.23 (2.44,7.33) <.001 Amp1q21 25/178 (14%) 3.56 (1.73, 7.31) <.001 3.10 (1.59,6.07) <.001 GEP IFN >= 11 55/227 (24%) 1.92 (1.02, 3.61) 0.044 1.76(1.00, 3.12) 0.050 GEP NFKB score >= 11 101/227 (44%) 0.89 (0.48, 1.64)0.705 0.90 (0.52, 1.55) 0.699 GEP Centrosome Index >= 3 86/227 (38%)3.89 (2.05, 7.37) <.001 2.84 (1.63, 4.93) <.001 GEP ProliferationIndex >= 10 28/227 (12%) 3.99 (2.09, 7.60) <.001 3.48 (1.93, 6.26) <.001GEP Poly PC Score >= 13 8/227 (4%) 1.31 (0.32, 5.45) 0.707 1.01 (0.24,4.15) 0.992 Multivariate 4 signals of 1q21 23/161 (14%) 2.38 (0.96,5.87) 0.061 2.66 (1.20, 5.91) 0.016 B2M > 5.5 mg/L 43/161 (27%) NS2 NS22.66 (1.34, 5.30) 0.005 GEP Centrosome Index >= 3 61/161 (38%) 2.95(1.27, 6.90) 0.012 NS2 NS2 GEP IFN >= 11 43/161 (27%) 2.85 (1.28, 6.36)0.010 2.87 (1.39, 5.90) 0.004 GEP Proliferation Index >= 10 16/161 (10%)3.13 (1.35, 7.29) 0.008 3.46 (1.59, 7.51) 0.002 HR—Hazard Ratio, 95%CI—95% Confidence Interval, P-value from Wald Chi-Square Test in CoxRegression NS2—Multivariate results not statistically significant at0.05 level. All univariate p-values reported regardless of significance.Multivariate model uses stepwise selection with entry level 0.1 andvariable remains if meets the 0.05 level. A multivariate p-value greaterthan 0.05 indicates variable forced into model with significantvariables chosen using stepwise selection.

TABLE 14c Univariate and multivariate associations of FISH-defineddel1p13 and molecular and clinical variables with overall and event-freesurvival in TT3 Overall Survival in TT3 Event-free survival TT3 Variablen/N (%) HR (95% CI) P-value HR (95% CI) P-value Univariate Age >= 65 yr68/235 (29%) 1.34 (0.71, 2.54) 0.366 1.36 (0.77, 2.39) 0.286 Albumin <3.5 g/dL 79/234 (34%) 2.33 (1.28, 4.27) 0.006 2.10 (1.24, 3.58) 0.006B2M >= 3.5 mg/L 123/233 (53%) 2.27 (1.20, 4.31) 0.012 2.21 (1.25, 3.88)0.006 B2M > 5.5 mg/L 61/233 (26%) 3.93 (2.14, 7.21) <.001 4.19 (2.45,7.16) <.001 Creatinine >= 2 mg/dL 17/234 (7%) 2.02 (0.79, 5.14) 0.1402.26 (1.02, 5.00) 0.044 Hb < 10 g/dL 80/234 (34%) 1.87 (1.03, 3.40)0.041 2.09 (1.23, 3.55) 0.006 LDH >= 190 U/L 64/234 (27%) 1.73 (0.93,3.21) 0.083 2.12 (1.24, 3.62) 0.006 CRP >= 8 mg/L 74/233 (32%) 1.24(0.66, 2.33) 0.496 1.12 (0.63, 1.96) 0.705 Cytogenetic Abnormalities97/231 (42%) 2.88 (1.53, 5.43) 0.001 2.34 (1.36, 4.02) 0.002 GEP HighRisk 41/223 (18%) 4.22 (2.24, 7.94) <.001 4.20 (2.41, 7.34) <.001del1p13 51/235 (22%) 1.55 (0.79, 3.01) 0.201 1.72 (0.97, 3.06) 0.062 GEPIFN >= 11 55/223 (25%) 1.91 (1.01, 3.61) 0.047 1.76 (0.99, 3.12) 0.053GEP NFKB score >= 11 100/223 (45%) 0.93 (0.50, 1.73) 0.822 0.92 (0.53,1.60) 0.779 GEP Centrosome Index >= 3 83/223 (37%) 3.38 (1.78, 6.38)<.001 2.55 (1.47, 4.44) <.001 GEP Proliferation Index >= 10 25/223 (11%)4.15 (2.14, 8.04) <.001 3.64 (1.99, 6.64) <.001 GEP Poly PC Score >= 138/223 (4%) 1.30 (0.31, 5.41) 0.716 1.00 (0.24, 4.13) 0.998 Multivariatedel1p13 47/216 (22%) 0.84 (0.39, 1.84) 0.669 0.97 (0.50, 1.89) 0.925B2M > 5.5 mg/L 57/216 (26%) NS2 NS2 2.44 (1.31, 4.55) 0.005 GEP HighRisk 40/216 (19%) 2.96 (1.33, 6.61) 0.008 3.16 (1.59, 6.28) 0.001 GEPCentrosome Index >= 3 80/216 (37%) 2.30 (1.07, 4.96) 0.033 NS2 NS2 GEPIFN >= 11 52/216 (24%) 2.57 (1.33, 4.96) 0.005 2.10 (1.16, 3.80) 0.015HR—Hazard Ratio, 95% CI—95% Confidence Interval, P-value from WaldChi-Square Test in Cox Regression NS2—Multivariate results notstatistically significant at 0.05 level. All univariate p-valuesreported regardless of significance. Multivariate model uses stepwiseselection with entry level 0.1 and variable remains if meets the 0.05level. A multivariate p-value greater than 0.05 indicates variableforced into model with significant variables chosen using stepwiseselection.

TABLE 14d Univariate and multivariate associations of FISH defineddel13q14 and molecular and clinical variables with overall andevent-free survival in TT3 Overall Survival in TT3 Event-free survivalTT3 Variable n/N (%) HR (95% CI) P-value HR (95% CI) P-value UnivariateAge >= 65 yr 71/243 (29%) 1.38 (0.74, 2.56) 0.312 1.42 (0.82, 2.48)0.211 Albumin < 3.5 g/dL 82/242 (34%) 2.08 (1.15, 3.75) 0.015 1.99(1.17, 3.37) 0.011 B2M >= 3.5 mg/L 124/241 (51%) 2.31 (1.24, 4.30) 0.0082.42 (1.38, 4.24) 0.002 B2M > 5.5 mg/L 63/241 (26%) 3.92 (2.17, 7.10)<.001 4.37 (2.57, 7.43) <.001 Creatinine >= 2 mg/dL 17/242 (7%) 1.98(0.78, 5.03) 0.149 2.30 (1.04, 5.09) 0.039 Hb < 10 g/dL 81/242 (33%)1.77 (0.99, 3.19) 0.055 2.09 (1.24, 3.53) 0.006 LDH >= 190 U/L 66/242(27%) 1.99 (1.10, 3.62) 0.024 2.47 (1.46, 4.18) <.001 CRP >= 8 mg/L78/241 (32%) 1.38 (0.75, 2.52) 0.298 1.26 (0.73, 2.18) 0.407 CytogeneticAbnormalities 103/239 (43%) 2.91 (1.56, 5.43) <.001 2.26 (1.31, 3.87)0.003 GEP High Risk 42/229 (18%) 4.21 (2.28, 7.79) <.001 4.43 (2.56,7.67) <.001 del13q14 121/243 (50%) 1.15 (0.64, 2.06) 0.646 1.38 (0.82,2.35) 0.229 GEP IFN >= 11 56/229 (24%) 1.80 (0.96, 3.37) 0.068 1.73(0.98, 3.06) 0.059 GEP NFKB score >= 11 104/229 (45%) 0.92 (0.50, 1.68)0.782 0.95 (0.55, 1.63) 0.856 GEP Centrosome Index >= 3 87/229 (38%)3.56 (1.90, 6.66) <.001 2.81 (1.62, 4.88) <.001 GEP ProliferationIndex >= 10 28/229 (12%) 3.87 (2.04, 7.35) <.001 3.52 (1.96, 6.34) <.001GEP Poly PC Score >= 13 8/229 (3%) 1.29 (0.31, 5.36) 0.724 1.02 (0.25,4.20) 0.978 Multivariate del13q14 111/222 (50%) 0.83 (0.44, 1.58) 0.5780.95 (0.53, 1.71) 0.870 B2M > 5.5 mg/L 59/222 (27%) NS2 NS2 2.53 (1.37,4.68) 0.003 Creatinine >= 2 mg/dL 16/222 (7%) 2.97 (1.10, 8.01) 0.032NS2 NS2 Cytogenetic Abnormalities 96/222 (43%) 2.81 (1.42, 5.55) 0.003NS2 NS2 GEP High Risk 41/222 (18%) NS2 NS2 3.32 (1.74, 6.33) <.001 GEPIFN >= 11 53/222 (24%) 2.60 (1.32, 5.11) 0.006 2.07 (1.14, 3.77) 0.017GEP Centrosome Index >= 3 84/222 (38%) 3.85 (1.93, 7.69) <.001 NS2 NS2GEP Poly PC Score >= 13 7/222 (3%)  6.32 (1.29, 30.92) 0.023 NS2 NS2HR—Hazard Ratio, 95% CI—95% Confidence Interval, P-value from WaldChi-Square Test in Cox Regression NS2—Multivariate results notstatistically significant at 0.05 level. All univariate p-valuesreported regardless of significance. Multivariate model uses stepwiseselection with entry level 0.1 and variable remains if meets the 0.05level. A multivariate p-value greater than 0.05 indicates variableforced into model with significant variables chosen using stepwiseselection.Prognostic and Predictive Impact of Genetic Lesions of IndividualChromosomes

Three, four, or greater copies of 1q21 were seen 188 of 517 (37%)tested. Greater than 4 copies of 1q21, present in 62 of 517 (12%) cases,but not three copies of 1q21, was a significant adverse feature for OSin all cases combined (P=0.0009) and in TT3 (P=0.001). While there was atrend for cases with 3 or 4 copies of 1q21 to have an inferior OS thancases lacking this abnormality in TT2-thalidomide (P=0.19) (FIG. 12C)this trend was not evident in TT2+thalidomide (P=0.89) (FIG. 12D). Thelack of a difference in OS in the TT2 samples may be due to small samplesize. Event-free survival is adversely affected in all cases combined,TT3 and TT2-thalidomide, but not in the TT2+thalidomide arm. Event freesurvival results were comparable, with disease with greater than 4copies of 1q21 exhibiting inferior survival when considering all cases,in TT3/TT3b (P=0.005) and TT2-thalidomide (P<0.0001), but notTT2+thalidomide (P=0.91) (FIG. 13A-D). Deletion of 1p13, present in 107of 517 (21%) cases tested, was significant poor risk factor whenconsidering OS (FIG. 14A-D) and EFS (FIG. 15A-D). Deletion of 1p13 wasnot adverse factor for OS and EFS in TT3. Remarkably, the presence ofdeletion of 1p13 was associated with a 3-year OS of 40% versus 80% inthose lacking deletion treated with TT2+thalidomide (P=0.01). Theadverse prognostic impact of 1p13 loss on in the TT2+thalidomide arm wasnot evident in the TT2-thalidomide arm (P=0.52). The EFS associationswere more pronounced. This was evident in of all cases (P=0.03).Although not significant, there is a clear trend for a negative impactof loss of 1p13 in TT3 (P=0.08). The negative impact of 1p13 deletion onEFS in the thalidomide arm was dramatic with median EFS of 12 monthscompared to 65 months for those not having 1p13 loss (P=0.04).Remarkably, the impact of 1p13 loss on outcome in the TT2-thalidomidearm was not evident, with median EFS for those with loss of 1p13 at 30months versus 40 months for those without loss of 1p13 (P=0.69). Takentogether, these data suggests that myeloma lacking 1p13 deletionbenefits from the addition of thalidomide while the addition ofthalidomide may have a negative impact on survival in those with diseaseharboring a loss of 1p13, and that bortezomib can partially overcomethis negative interaction. Chromosome 13q14 deletion was found in 2457of 517 (48%) of cases. A somewhat surprising finding was the clear lackof prognostic significance of chromosome 13q14 deletion in any of theanalyses performed for OS or EFS in all trials (FIGS. 16A-D and 17A-D).

Prognostic and Predictive Implications of Combinations of GeneticLesions

With strong evidence that the 70-gene high risk GEP signature, seen inapproximately 14% of newly diagnosed disease, is driven by copynumber-dependent elevated expression of genes mapping to 1q21 andreduced expression of genes mapping to chromosome 1p, disease withTRI-FISH-defined gain of 1q21 and loss of 1p13 was tested to see ifthere is an association with inferior survival. Disease with three ormore copies of 1q21 and concomitant loss of one copy of 1p13 was seen in38 of 517 (7%) of cases tested (FIG. 18A-D). The 3-year OS for all caseswith three or more copies of 1q21 and 1 copy of 1p13 was approximately40% compared to 80% in those with no more than one of theseabnormalities (P<0.0001) (FIG. 18A-D). Present in 13 of 234 (5%) casestreated with TT3, this constellation was associated with a 3-year OS of55% versus 83% in those with at most one of these abnormalities(P=0.005) (FIG. 18A-D). Of 61 patients treated on the TT2+thalidomidearm these two abnormalities were concurrent in 6 (10%). The 3-year OSfor this group was 20% relative to 83% for cases have at most one ofthese abnormalities (P=0.0002) (FIG. 9). The number of cases in theTT2-thalidomide arm was too small to provide definitive results at thistime. The 3-year EFS for all cases with three or more copies of 1q21 and1 copy of 1p13 was 30% versus 70% for those with at most one abnormality(P<0.0001) (FIG. 19A-D). This value was 55% versus 80% in TT3 (P=0.03),0% versus 60% in TT2-thalidomide (P=0.06) and 20% versus 70% inTT2-thalidomide (P=0.04). Taken together these data suggest that currenttherapies are not providing significant improvement in outcome forpatients with disease harboring gains of 1q21 and loss of 1p13.

When considering all cases, 3 or 4 signals for 1q21 and 1 signal for13q14 were observed in 124 of 517 (24%) tumors. This constellation ofwas associated with shorter EFS times than those with no abnormalitiesin these two chromosomes or abnormalities in only one chromosome(P=0.0003) (FIG. 20A-D). Present in 55 of 247 (22%) this geneticcombination was not associated with an inferior EFS (P=0.13) in TT3samples. Moreover, this combination was not an adverse prognostic factorin TT2+thalidomide (P=0.74), but represented a powerful poor riskfeature in TT2-thalidomide (P<0.0001), with the 25% of cases with tumorshaving both abnormalities experiencing a median EFS of 24 months versus48 months for those with one or none of these two genetic lesions.Although similar trends were observed for OS, the presence of bothlesions was only significant when considering all cases (P=0.004) (FIG.21A-D). There was a borderline significance for the TT2-thalidomidegroup (P=0.06), which may emerge as significant as more samples aretested.

Thus, in summary, fluorescence in situ hybridization (FISH) analysis for1q21 amplification and deletion 13 represents a better method for riskassessment than any other that exists. Additionally, since FISHdemonstrated that the strong correlation of expression of CKS1B withgene copy number and also that the risk of death increased with increasein gene copy number, FISH could be used to detect residual disease andpredict recurrence and progression. Since Western blot analysisdemonstrated that an increase in the levels of CKS1B protein, CKS1B orany pathway in which it works could be therapeutically targeted. Thus,the amplification of 1q21 could be a diagnostic, prognostic andpotential target in cancer especially myeloma.

The following references are cited herein:

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Any patents or publications mentioned in this specification areindicative of the levels of those skilled in the art to which theinvention pertains. Further, these patents and publications areincorporated by reference herein to the same extent as if eachindividual publication was specifically and individually indicated to beincorporated by reference.

What is claimed is:
 1. A method of detecting the presence of a multiplemyeloma 3 (MM3) or multiple myeloma 4 (MM4) subtype myeloma cell in amultiple myeloma patient, comprising: (a) detecting the gene expressionlevel of one or more interferon-induced genes in a biological samplecomprising CD138+ plasma cells obtained from the patient by isolatingRNA isolated from the plasma cells and contacting the isolated RNA witha nucleic acid probe specific for each of the one or moreinterferon-induced genes, wherein the one or more interferon-inducedgenes is selected from among IF127, MX1, MX2, OAS1, OAS2, IFIT1, IFIT4,PLSCR1, and STAT1, and (b) detecting the presence of an MM3 or MM4subtype myeloma cell if the gene expression level of the one or moreinterferon-induced genes is increased relative to the gene expressionlevel in a sample of normal CD138+ plasma cells.
 2. The method of claim1, wherein the one or more interferon-induced genes comprise STAT1. 3.The method of claim 1, wherein the patient is undergoing treatment withbortezomib.
 4. The method of claim 1, wherein the patient is undergoingtreatment with thalidomide.
 5. The method of claim 1, wherein thebiological sample comprises one or more of tonsil tissue, bone marrowtissue, mucosal tissue, lymph node tissue or peripheral blood.
 6. Themethod of claim 1, further comprising separating the CD138+ plasma cellsfrom other cells in the biological sample prior to RNA isolation.
 7. Themethod of claim 6, wherein separating the CD138+ plasma cells comprisesanti-CD138 immunomagnetic bead selection.
 8. The method of claim 1,wherein the nucleic acid probe specific for each of the one or moreinterferon-induced genes is part of a DNA microarray.
 9. The method ofclaim 1, wherein the method comprises real time polymerase chainreaction (RT-PCR).
 10. The method of claim 1, wherein the isolated RNAis reverse transcribed into cDNA.
 11. The method of claim 1, furthercomprising obtaining the biological sample from the patient.
 12. Themethod of claim 1, further comprising administering a treatment forhigh-risk multiple myeloma if an MM3 or MM4 subtype myeloma cell isdetected.
 13. The method of claim 1, wherein the one or moreinterferon-induced genes comprise IF127 and STAT1.